Centroid markers for image analysis of high density clusters in complex polynucleotide sequencing

ABSTRACT

Improved compositions, methods, apparatus, and kits for high-throughput nucleic acid amplification, detection and sequencing are disclosed. A nucleic acid cluster having an identifiable center is produced by generating on a solid support an immobilized nucleic acid complement from a template, one of which comprises a detectable label; and amplifying the complement and the template to obtain a nucleic acid cluster on the support, the cluster having a substantially central location marked by the detectable label and a surrounding region comprising immobilized copies. Also disclosed are nucleotide sequence determination in nucleic acid clusters so produced, center position annotation in the clusters, assignment of sequence information to overlapping clusters, and related compositions and methods.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 13/445,072,filed Apr. 12, 2012, now U.S. Pat. No. 8,541,172, which is acontinuation of U.S. application Ser. No. 12/559,902, filed Sep. 15,2009, each of which is incorporated herein by reference in its entirety.

BACKGROUND

1. Technical Field

The present invention generally relates to the field of nucleic acidamplification, detection and sequencing. More specifically, the presentinvention relates to improved compositions, methods, apparatus, and kitsfor high-throughput nucleic acid amplification, detection andsequencing.

2. Description of the Related Art

High-throughput technologies have become a cornerstone in many areas ofmodern molecular biology, biotechnology and medicine. For example,efforts to rapidly, accurately, and economically determine geneexpression levels (e.g., microarrays) and nucleic acid sequence (e.g.,parallel sequencing) have intensified over the past few years. Theinformation provided by such advances has furthered genome analyses forseveral plant and animal species, including humans, non-human primatesand others, and has also assisted drug target discovery and validation,disease diagnosis and risk scoring, and the identification andcharacterization of multiple organisms.

A number of methods of nucleic acid sequencing are well known anddocumented in the art. The two most commonly used methods are the Maxamand Gilbert technique and the widely used Sanger sequencing technique.

In Sanger sequencing, each nucleic acid molecule to be sequenced is usedas a template that is replicated in a reaction employing DNA polymeraseas a catalytic enzyme, and deoxynucleotide triphosphates (dNTPs) ATP,CTP, GTP and TTP as precursors to be incorporated into a DNA complementof the template and dideoxynucleotide triphosphates of adenine (A),guanine (G), cytosine (C) and thymidine (T) (ddNTPs) as chainterminators. The DNA polymerase can incorporate both dNTPs and ddNTPsinto the growing DNA strand. The incorporation of a ddNTP, however,terminates the nucleic acid chain extension because the ddNTP lacks a 3′hydroxyl group and thus is no longer a substrate for further chainelongation. For example, in a particular template-directed Sangersequencing reaction in which only one type of ddNTP (e.g., ddCTP) ispresent, a mixture of nucleic acids of different lengths is produced,all terminating with the same ddNTP (e.g., ddCTP). Typically, eitherseparate reactions are set up for each of the four types of ddNTPs orthe four ddNTPs are differentially labeled and used in a singlereaction, and size distribution of the nucleic acid fragment products isanalyzed by denaturing gel electrophoresis or by mass spectrometry. Forexample, each of the ddNTPs in the reaction mixture is labeled with adifferent fluorophore to enable detection of the fragments of differentlengths.

The above described methods are disadvantageous because each nucleicacid to be sequenced has to be processed individually during thesequencing reaction. Gel electrophoresis is not well suited for largescale high throughput sequencing. It is cumbersome, labor intensive, andintrinsically slow, even when capillary gel electrophoresis is used. Inaddition, following electrophoretic separation of reaction products, thesubsequent analysis of electrophoretograms for determination of thesequence is time-consuming and can generate equivocal results due toconfounding artifacts. Mass spectrometry offers more promise forexpediting sequence determination, but it is still at the prototypelevel, requires very expensive apparatus and labor-intensive instrumentmaintenance, and each sample must be analyzed individually.

More recently, nucleic acid sequencing methods based on solid-phase DNAchips and DNA hybridization have become available. Each of these methodsis not without its shortcomings, however, because DNA chips have to becarefully designed, fastidiously manufactured, and subjected to rigorousquality control testing. These processes are lengthy and requiresignificant expertise, which drives up the price of individual chips.Moreover, often the chips are not reusable and thus for each chip, onlyone nucleic acid sample (e.g., one patient to be diagnosed) can beprocessed at a time.

In many currently practiced techniques for nucleic acid sequenceanalysis, amplification of the nucleic acids of interest is aprerequisite step in order to obtain the nucleic acid in a quantitysufficient for analysis. Several methods of nucleic acid amplificationare well known and documented in the art. For example, nucleic acids canbe amplified by inserting a nucleic acid of interest into an expressionvector construct. Such vectors can then be introduced into suitablebiological host cells and the vector DNA, including the nucleic acid ofinterest, is amplified by culturing the biological host using wellestablished protocols. However, such methods have the disadvantage ofbeing time consuming, labor intensive, and difficult to automate.

The technique of DNA amplification by the polymerase chain reaction(PCR) is a widely used and well documented method. In PCR, a targetnucleic acid fragment of interest can be amplified using one or twoshort oligonucleotide sequences (usually referred to as primers) thatspecifically hybridize (e.g., by Watson-Crick base-pairing) to knownsequences flanking the DNA sequence that is to be amplified. By repeatedcycles of heat denaturation, primer hybridization, and extension, thetarget nucleic acid is exponentially amplified. Traditionally, thismethod is performed in solution and the amplified target nucleic acidfragment is purified from solution by methods well known in the art, forexample, by gel electrophoresis.

More recently, nucleic acid amplification methods have been disclosedwhich employ an immobilized primer grafted to a solid-phase surface inconjunction with free primers in solution. These methods allow thesimultaneous amplification and attachment of a PCR product onto thesurface.

Some known methods of nucleic acid analysis involve PCR-basedamplification of a target nucleic acid only when the target nucleic acidis present in the sample being tested. For the amplification of thetarget sequence, primers can be attached to a solid support, whichresults in the amplified target nucleic acid sequences also beingattached to the solid support. This amplification technique is oftenreferred to as the “bridge amplification” technique. In this technique,conventional PCR primers can be designed to hybridize specifically topolynucleotide sequences flanking the particular target nucleic acidsequence to be amplified. If the target nucleic acid is present in thesample, it hybridizes to the primers and is amplified by PCR. The firststep in this PCR amplification process is thus the hybridization of thetarget nucleic acid to a first specific primer attached to the support(“primer 1”). A first amplification product, which is complementary tothe target nucleic acid, is then formed by extension of the primer 1sequence. Denaturation conditions release the target nucleic acid, whichcan then either participate in further hybridization reactions withother primer 1 sequences attached to the support or be removed from thesolid support. The first amplification product, which is attached to thesupport, can then hybridize with a second specific primer (“primer 2”)attached to the support and a second amplification product comprising anattached nucleic acid sequence complementary to the first amplificationproduct can be formed by extension of the primer 2 sequence. Thus, thetarget nucleic acid and the first and second amplification products arecapable of participating in a plurality of hybridization and extensionreactions, which are limited by the initial presence or absence of thetarget nucleic acid and by the number of primer 1 and primer 2 sequencesinitially attached to the solid support.

A bridge amplification technique can be used to amplify severaldifferent target nucleic acid sequences simultaneously by arrayingdifferent sets of first and second primers, each set being specific fora different target nucleic acid sequence, on different or overlappingregions of the solid support. A further application of the bridgeamplification technique is to amplify fragments using immobilizedprimers which are complementary to a universal sequence located at theends of a collection of templates of different sequence. Thus the primer1 and primer 2 sequences may be complementary to a nucleic acid samplewith known ends, for example, ends that have been attached to the sampleby ligation of a universal adapter sequence. The templates may beapplied to the solid support as single strands where the ends of eachstrand are complementary to, and hybridize with, the primer 1 and/orprimer 2 sequences. Primer 1 can be extended to form an extensionproduct where the end of the extension product is complementary to theprimer 2 sequence. Likewise, primer 2 can be extended to form anextension product where the end of the extension product iscomplementary to the primer 1 sequence. The hybridized targets can bedenatured and removed from the support. The first extension products canbe hybridized with the primer 1 and primer 2 sequences and extended toform second extension products wherein the second extension products arecomplementary copies of the first extension products. The first andsecond extension products can be amplified via cycles of denaturation,hybridization and extension to produce multiple copies of each of thefirst and second extension products. The amplification may give rise toa population of nucleic acid clusters attached to the support where eachcluster is derived from a single template, but adjacent clusters on thesolid support contain different template sequences.

In the era of high-throughput technology, amassing the highest yield ofinterpretable data at the lowest cost per effort remains a significantchallenge. Cluster-based methods of nucleic acid sequencing, such asthose that utilize bridge amplification for cluster formation, have madea valuable contribution toward the goal of increasing the throughput ofnucleic acid sequencing. These cluster-based methods rely on sequencinga dense population of nucleic acids immobilized on a solid support, andtypically involve the use of image analysis software to deconvoluteoptical signals generated in the course of simultaneously sequencingmultiple clusters situated at distinct locations on a solid support.

However, such solid-phase nucleic acid cluster-based sequencingtechnologies still face considerable obstacles that limit the amount ofthroughput that can be achieved. For example, in cluster-basedsequencing methods, determining the nucleic acid sequences of two ormore clusters that are physically too close to one another to beresolved spatially, or that in fact physically overlap on the solidsupport, can pose an obstacle. For example, current image analysissoftware can require valuable time and computational resources fordetermining from which of two overlapping clusters an optical signal hasemanated. As a consequence, compromises are inevitable for a variety ofdetection platforms with respect to the quantity and/or quality ofnucleic acid sequence information that can be obtained.

High density nucleic acid cluster-based genomics methods extend to otherareas of genome analysis as well. For example, nucleic acidcluster-based genomics can be used in sequencing applications,diagnostics and screening, gene expression analysis, epigeneticanalysis, genetic analysis of polymorphisms, and the like. Each of thesenucleic acid cluster-based genomics technologies, too, is limited whenthere is an inability to resolve data generated from closely proximateor spatially overlapping nucleic acid clusters.

Clearly there remains a need for increasing the quality and quantity ofnucleic acid sequencing data that can be obtained rapidly andcost-effectively for a wide variety of uses, including for genomics(e.g., for genome characterization of any and all animal, plant,microbial or other biological species or populations), pharmacogenomics,transcriptomics, diagnostics, prognostics, biomedical risk assessment,clinical and research genetics, personalized medicine, drug efficacy anddrug interactions assessments, veterinary medicine, agriculture,evolutionary and biodiversity studies, aquaculture, forestry,oceanography, ecological and environmental management, and otherpurposes. The presently disclosed invention embodiments providecompositions and methods that address these and similar needs, includingcompositions and methods to increase the level of throughput inhigh-throughput nucleic acid sequencing technologies, and offer otherrelated advantages. These and other aspects of the present inventionwill become apparent upon reference to the following detaileddescription.

BRIEF SUMMARY

According to certain embodiments of the present invention there isprovided a method for producing at least one nucleic acid cluster havingan identifiable center, comprising (I) generating, on a solid support,at least one immobilized nucleic acid complement of at least one nucleicacid template, wherein the at least one nucleic acid template or the atleast one nucleic acid complement comprises a detectable label; and (II)amplifying the at least one nucleic acid template and the at least onenucleic acid complement to obtain on the solid support at least onenucleic acid cluster, wherein each cluster comprises (a) a substantiallycentral location comprising the at least one nucleic acid template andthe at least one nucleic acid complement, and (b) a surrounding regioncomprising one or more immobilized copies of the at least one nucleicacid template and of the at least one nucleic acid complement, andthereby producing the at least one nucleic acid cluster having anidentifiable center.

In certain further embodiments the one or more immobilized copies of theat least one nucleic acid template and the at least one nucleic acidcomplement at the surrounding region lack the detectable label, andwhereby the detectable label distinguishes the substantially centrallocation from the surrounding region. In certain embodiments at least100 nucleic acid clusters each having an identifiable center areproduced per square centimeter on the solid support. In certainembodiments the step of amplifying is repeated one or a plurality oftimes under conditions and for a time sufficient for the detectablelabel to remain at the substantially central location in the at leastone nucleic acid cluster. In certain embodiments the at least onenucleic acid template is immobilized via its 5′ end to the solidsupport. In certain embodiments step (I) comprises generating at leastone immobilized detectably labeled central nucleic acid complement ofthe at least one nucleic acid template, on the solid support, whereinthe solid support comprises one or more immobilized oligonucleotideprimers X, and wherein (a) the at least one nucleic acid template has a5′ end and a 3′ end and comprises an oligonucleotide sequence Y at the5′ end and an oligonucleotide sequence Z at the 3′ end, (b) each of theone or more immobilized oligonucleotide primers X (i) is immobilized atits 5′ end to the solid support and (ii) is capable of hybridizing tothe oligonucleotide sequence Z, and (c) the at least one immobilizeddetectably labeled central nucleic acid complement comprises (i) anextension of one of the immobilized oligonucleotide primers X, and (ii)at least one detectable label. In certain further embodiments theoligonucleotide sequence Z is complementary to the oligonucleotidesequence Y and the immobilized oligonucleotide primer X comprises asequence that is substantially identical to oligonucleotide sequence Y.In certain other further embodiments the solid support comprises a firstand a second immobilized oligonucleotide primer X which are differentfrom each other, wherein the oligonucleotide sequence Z can hybridize tothe first immobilized oligonucleotide primer X and the secondimmobilized oligonucleotide primer X has a sequence that issubstantially identical to oligonucleotide sequence Y. In certain otherfurther embodiments generating comprises (a) initiating, in the presenceof one or more labeled nucleotides that each comprise a detectablelabel, a primer extension reaction on the at least one nucleic acidtemplate whereby the one or more immobilized oligonucleotide primers Xare extended by incorporation, into the immobilized detectably labeledcentral nucleic acid complement, of one or more of said labelednucleotides that each comprise a detectable label; (b) stopping theprimer extension reaction and removing therefrom unincorporated labelednucleotides; and (c) re-initiating the primer extension reaction in thepresence of unlabeled nucleotides. In certain further embodiments steps(a) and (b) are repeated one or a plurality of times, wherein a singlenucleotide comprising a detectable label is incorporated into thecentral nucleic acid complement at each step (a).

According to certain of the herein described embodiments, amplifyingcomprises thermocycling amplification, and in certain embodimentsamplifying comprises isothermal amplification.

According to certain embodiments the herein described method furthercomprises recording a center position in the nucleic acid cluster bydetecting the at least one detectable label and therefrom identifyingthe substantially central location in the cluster. In certain furtherembodiments the method comprises determining a nucleotide sequence inthe at least one nucleic acid cluster by performing one or a pluralityof sequencing steps on at least one of (i) the at least one nucleic acidtemplate or an immobilized copy thereof, and (ii) the immobilizeddetectably labeled central nucleic acid complement or an immobilizedcopy thereof. In a further embodiment each sequencing step comprisesincorporating a labeled sequencing nucleotide into the at least onenucleic acid cluster; and subsequently detecting the incorporatedlabeled sequencing nucleotide in the at least one nucleic acid cluster.In certain other further embodiments, full or partial nucleotidesequences are determined simultaneously in more than one nucleic acidcluster, each of the more than one clusters having an identifiedsubstantially central location. According to certain relatedembodiments, (i) full or partial nucleotide sequences are determinedsimultaneously in more than one nucleic acid cluster, each of said morethan one clusters having an identifiable center, (ii) each sequencingstep further comprises detecting a position of the incorporated labeledsequencing nucleotide on the solid support, and (iii) a determinedsequence can be assigned to a discrete nucleic acid cluster when thereis a substantial correlation between (i) the recorded center position ofeach nucleic acid cluster and (ii) the position of each incorporatedlabeled sequencing nucleotide on the solid support that is detected ineach sequencing step. In certain further embodiments, for each of two ormore overlapping nucleic acid clusters, the determined nucleotidesequence is assigned to one discrete nucleic acid cluster.

According to certain embodiments of the above described methods, thestep of recording is performed prior to amplifying of step (II), and incertain other embodiments the step of recording is performed afteramplifying of step (II). In certain other embodiments the one or moredetectable labels are not detectably discrete from any incorporatedlabeled sequencing nucleotide detected at each sequencing step. Incertain embodiments the step of recording is performed at eachsequencing step. In certain embodiments the one or more detectablelabels are detectably discrete from each of the incorporated labeledsequencing nucleotides detected at each sequencing step.

In certain other embodiments of the present invention there is provideda method for annotating a center position of at least one nucleic acidcluster, comprising: (I) generating, on a solid support, at least oneimmobilized nucleic acid complement of at least one nucleic acidtemplate, wherein the at least one nucleic acid template or the at leastone nucleic acid complement comprises a detectable center label; and(II) amplifying the at least one nucleic acid template and the at leastone nucleic acid complement to obtain on the solid support at least onenucleic acid cluster, wherein each cluster comprises (a) a substantiallycentral location comprising the at least one nucleic acid template andthe at least one nucleic acid complement, and (b) a surrounding regioncomprising one or more immobilized copies of the at least one nucleicacid template and of the at least one nucleic acid complement; and (III)recording a center position in the at least one nucleic acid cluster bydetecting the detectable center label to identify the substantiallycentral location in the at least one nucleic acid cluster as the centerposition, and thereby annotating the center position of the at least onenucleic acid cluster.

In certain further embodiments the one or more immobilized copies of theat least one nucleic acid template and the at least one nucleic acidcomplement at the surrounding region lack the detectable center label,and whereby the detectable center label distinguishes the substantiallycentral location from the surrounding region. In another embodiment atleast 100 nucleic acid clusters each having an annotated center positionare produced per square centimeter on the solid support. In anotherembodiment the step of amplifying is repeated one or a plurality oftimes under conditions and for a time sufficient for the detectablecenter label to remain at the substantially central location in the atleast one nucleic acid cluster. In another embodiment the at least onenucleic acid template is immobilized via its 5′ end to the solidsupport. In another embodiment step (I) comprises generating at leastone immobilized detectably labeled central nucleic acid complement ofthe at least one nucleic acid template, on the solid support, whereinthe solid support comprises one or more immobilized oligonucleotideprimers X, and wherein (a) the at least one nucleic acid template has a5′ end and a 3′ end and comprises an oligonucleotide sequence Y at the5′ end and an oligonucleotide sequence Z at the 3′ end, (b) each of theone or more immobilized oligonucleotide primers X (i) is immobilized atits 5′ end to the solid support and (ii) is capable of hybridizing tothe oligonucleotide sequence Z, and (c) the at least one immobilizeddetectably labeled central nucleic acid complement comprises (i) anextension of one of the immobilized oligonucleotide primers X, and (ii)at least one detectable label. In certain further embodiments theoligonucleotide sequence Z is complementary to the oligonucleotidesequence Y and the immobilized oligonucleotide primer X comprises asequence that is substantially identical to oligonucleotide sequence Y.In certain other further embodiments the solid support comprises a firstand a second immobilized oligonucleotide primer X which are differentfrom each other, wherein the oligonucleotide sequence Z can hybridize tothe first immobilized oligonucleotide primer X and the secondimmobilized oligonucleotide primer X has a sequence that issubstantially identical to oligonucleotide sequence Y. In certain otherfurther embodiments, the step of generating comprises (a) initiating, inthe presence of one or more labeled nucleotides that each comprise adetectable center label, a primer extension reaction on the at least onenucleic acid template whereby the one or more immobilizedoligonucleotide primers X are extended by incorporation, into the atleast one immobilized detectably labeled central nucleic acidcomplement, of one or more of said labeled nucleotides that eachcomprise a detectable center label; (b) stopping the primer extensionreaction and removing therefrom unincorporated labeled nucleotides; and(c) re-initiating the primer extension reaction in the presence ofunlabeled nucleotides. In certain embodiments steps (a) and (b) arerepeated one or a plurality of times, and wherein a single nucleotidecomprising a detectable center label is incorporated into the at leastone central nucleic acid complement at each step (a).

In certain other further embodiments there is provided a methodcomprising determining a nucleotide sequence in the at least one nucleicacid cluster by performing one or a plurality of sequencing steps on atleast one of (i) the at least one nucleic acid template or animmobilized copy thereof, and (ii) the at least one immobilizeddetectably labeled central nucleic acid complement or an immobilizedcopy thereof. In a further embodiment each sequencing step comprisesincorporating a labeled sequencing nucleotide into the at least onenucleic acid cluster; and subsequently detecting said incorporatedlabeled sequencing nucleotide in the at least one nucleic acid cluster.In another further embodiment full or partial nucleotide sequences aredetermined simultaneously in more than one nucleic acid cluster, eachhaving an identified center position. In certain related embodiments,(i) full or partial nucleotide sequences are determined simultaneouslyin more than one nucleic acid cluster, each having an identified centerposition, (ii) each sequencing step further comprises detecting alabeled sequencing nucleotide position of the incorporated labeledsequencing nucleotide on the solid support, and (iii) a determinedsequence can be assigned to a discrete nucleic acid cluster when thereis a substantial correlation between (i) the recorded center position ofeach of the more than one nucleic acid clusters and (ii) the labeledsequencing nucleotide position of each incorporated labeled sequencingnucleotide on the solid support that is detected in each sequencingstep. In certain embodiments, for each of two or more overlappingnucleic acid clusters, the determined nucleotide sequence is assigned toone discrete nucleic acid cluster.

In certain embodiments of the herein described methods, the step ofrecording is performed prior to amplifying of step (II), and in certainembodiments the step of recording is performed after amplifying of step(II). In certain embodiments the at least one detectable center label isnot detectably discrete from any incorporated labeled sequencingnucleotide that is detected at each sequencing step. In certainembodiments the step of recording is performed at each sequencing step.In certain embodiments the step of recording is performed at eachsequencing step and the at least one detectable center label isdetectably discrete from each of the incorporated labeled sequencingnucleotides detected at each sequencing step. In certain embodiments thestep of amplifying is selected from a step that comprises thermocyclingamplification and a step that comprises isothermal amplification.

In certain embodiments of the present invention there is provided amethod for assigning nucleotide sequence information to at least one oftwo or more nucleic acid clusters that overlap on a solid support,comprising (a) providing two or more nucleic acid clusters thatdetectably overlap on a solid support, each of said clusters comprising(i) a substantially central location comprising an immobilized centralnucleic acid having a detectable center label, and (ii) a surroundingregion comprising one or more immobilized copies of the immobilizedcentral nucleic acid, wherein the detectable center label distinguishesthe substantially central location from the surrounding region; (b)detecting the detectable center label in each of said overlappingnucleic acid clusters and therefrom identifying in each cluster a firstposition for the substantially central location on the solid support;(c) determining a nucleotide sequence in at least one cluster of saidoverlapping nucleic acid clusters by performing one or a plurality ofsequencing steps thereupon, thereby identifying a second position forthe at least one cluster on the solid support; and (d) assigning anucleotide sequence determined in (c) to a discrete nucleic acid clusterby substantially correlating (i) the first position for thesubstantially central location of (b) with (ii) the second position forthe at least one cluster on the solid support of (c), and therebyassigning nucleotide sequence information to at least one of two or morenucleic acid clusters that overlap on the solid support.

In certain further embodiments each sequencing step comprises (i)incorporating one or more detectably labeled sequencing nucleotides intothe nucleic acid cluster, and (ii) detecting a second position on thesolid support for each of the one or more incorporated detectablylabeled sequencing nucleotides. In certain other further embodiments,two, three, four, five, or six overlapping nucleic acid clusters areeach assigned to a discrete nucleic acid cluster. In certain otherfurther embodiments, each detectable center label is not detectablydiscrete from any incorporated labeled sequencing nucleotide detected ateach sequencing step. In certain other embodiments the step of recordingis performed for each sequencing step. In certain further embodiments,each detectable center label is detectably discrete from anyincorporated labeled sequencing nucleotide detected at each sequencingstep.

In other embodiments there is provided by the present invention acomposition comprising (a) a solid support; and (b) one or more nucleicacid clusters each comprising a plurality of nucleic acids having thesame sequence and being immobilized to the solid support, wherein eachnucleic acid cluster comprises an identifiable center comprising a firstsubpopulation of the plurality of nucleic acids that is surrounded by asecond subpopulation of the nucleic acids, wherein each of the nucleicacids in the first subpopulation comprises one or more detectable centerlabels that distinguish the nucleic acids in the first subpopulationfrom the nucleic acids in the second population. In certain embodimentsthe composition comprises one or more oligonucleotide primers X. Incertain embodiments the nucleic acids of (i) and (ii) are singlestranded. In certain embodiments the nucleic acid clusters arecovalently immobilized to the solid support by a chemically modifiablefunctional group. In certain further embodiments the chemicallymodifiable functional group is selected from a phosphate group, acarboxylic moiety, an aldehyde moiety, a thiol, a hydroxyl, adimethoxytrityl (DMT) and an amino group. In certain embodiments thechemically modifiable functional group comprises an amino group. Incertain embodiments of the herein described composition, the solidsupport to which the 5′ ends are immobilized comprises a support that isselected from optical fibers, latex beads, dextran beads, polystyrene,polypropylene, a polyacrylamide gel, gold surfaces, glass surfaces andsilicon wafers. In certain embodiments the solid support is glass. Incertain embodiments of the herein described composition, the density ofeach of the one or more nucleic acid clusters on the solid support is10,000/mm² to 100,000/mm². In certain embodiments of the hereindescribed composition, the diameter of each of the one or more nucleicacid clusters is about 0.2 micrometers to about 6 micrometers. Incertain embodiments of the herein described composition, the diameter ofeach of the one or more nucleic acid clusters is about 0.5 micrometersto about 3 micrometers. In certain embodiments of the herein describedcomposition, at most 50 percent of the copies of the detectably labeledcentral nucleic acid complement comprise one or more detectable centerlabels. In certain embodiments of the herein described composition, atmost 5 percent of the copies of the detectably labeled central nucleicacid complement comprise one detectable center label. In certainembodiments of the herein described composition, each nucleic acidcluster comprises one detectably labeled central nucleic acidcomplement. In certain further embodiments the detectably labeledcentral nucleic acid complement comprises one or more detectable centerlabels. In certain still further embodiments the detectably labeledcentral nucleic acid complement comprises one detectable center label.

These and other aspects and embodiments of the invention will be evidentupon reference to the following detailed description and attacheddrawings. All of the U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference in their entirety, as if each was incorporated individually.Aspects of the invention can be modified, if necessary, to employconcepts of the various patents, applications and publications toprovide yet further embodiments of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows closely packed nucleic acid clusters on a surface in thefollowing configurations: FIG. 1A shows the cluster configuration usingopen circles to indicate cluster locations; FIG. 1B shows arepresentation of an image that would be produced if all of the nucleicacids in the clusters are labeled; FIG. 1C shows the clusterconfiguration with the locations of cluster centers indicated by a blackdot; FIG. 1D shows a representation of an image that would be producedif only nucleic acids located at a central location of each cluster arelabeled; FIG. 1E shows a representation of an image that would beproduced if nucleic acids located at a central location of each clusterlack a label that is present on nucleic acids surrounding each centrallocation.

FIG. 2 shows an embodiment of the invention used to label the center ofa nucleic acid cluster. A template is hybridized to a surface (FIG. 2A);the hybridized template is extended using labeled nucleotides to make anucleic acid complement (FIG. 2B); and the nucleic acid complement isamplified using unlabelled nucleotides to produce a cluster with anidentifiable center (FIG. 2C). The strands of adjacent clusters mayoverlap on the surface, but the initial strands remain discrete, and cantherefore be used to locate the adjacent clusters of different sequence.

FIG. 3 shows an image of an array of clusters where the center of thecluster is unlabeled, the labeled surrounding region forming a ring offluorescence around the unlabeled center. The center nucleic acids ofthe cluster were formed by extension using dUTP rather than dTTP. Thenucleic acids containing dUTP were digested using Uracil DNA glyosylase,and hence the cluster appears as a ring rather than a sphere, as thecentrally located strands were no longer present and hence were notcapable of being labeled.

DETAILED DESCRIPTION

The present invention is directed in certain embodiments as describedherein to a solid-phase immobilized nucleic acid cluster having anidentifiable center, to methods for producing such a nucleic acidcluster, and to a method for annotating a center position in such acluster. These and related embodiments will find uses in improving thequality and quantity of nucleic acid sequence information that can beobtained from a nucleic acid sample such as a nucleic acid template orits complement as described herein, for instance, a DNA or RNApolynucleotide or other nucleic acid sample. Accordingly, certainembodiments as disclosed herein may provide higher throughputpolynucleotide sequencing, for instance, higher rates of collection ofDNA or RNA sequence data, greater efficiency in sequence datacollection, and/or lower costs of obtaining such sequence data, relativeto previously available methodologies.

Certain embodiments as disclosed herein for the first time are based inpart on heretofore unappreciated advantages that are obtained byproducing a nucleic acid cluster having an identifiable center. Forexample, a center-labeled nucleic acid cluster can be provided bygenerating, on a solid support, an immobilized nucleic acid complementof a nucleic acid template such that at least one of the template andthe complement comprises a detectable label; and amplifying the templateand the complement to obtain a solid-phase immobilized nucleic acidcluster having (i) a substantially central location in which thedetectable label is present to provide an identifiable center, and (ii)a surrounding region comprising immobilized copies of the template andof the complement.

Alternatively, a nucleic acid having an identifiable center can beprovided by generating, on a solid support, an immobilized nucleic acidcomplement of a nucleic acid template such that at least one of thetemplate and the complement lacks a particular detectable label; andamplifying the template and the complement to obtain a solid-phaseimmobilized nucleic acid cluster having (i) a substantially centrallocation in which the detectable label is absent, and (ii) a surroundingregion comprising immobilized copies of the template and of thecomplement that have the particular detectable label to provide anidentifiable center. Thus, the cluster may in these and relatedembodiments appear to have a ring or donut shape, and the center can bedistinguished due to the absence of a detectable label that is presentin the surrounding region.

Without wishing to be bound by theory, these and related embodiments arebelieved to relate in part to unexpected advantages associated with anucleic acid cluster that results from radially outward growth of thecluster in all directions from the identifiable and substantiallycentral location at which the initial generation of the complement ofthe template occurs. Accordingly, and surprisingly, such embodimentsadvantageously afford a nucleic acid cluster having an identifiablecenter, for example, by virtue of the detectable label serving as abeacon that marks the center of the cluster (e.g., a detectable centerlabel as provided herein). Hence, where, prior to the presentdisclosure, a solid-phase immobilized nucleic acid cluster as providedherein, grown in a substantially symmetrical and progressively outwardmanner from a specifically identified substantially central originatinglocation, was neither contemplated nor predicted to confer anyparticular benefit, there is advantageously described herein for thefirst time a method for producing a nucleic acid cluster having anidentifiable center, along with methods for using such a center-labelednucleic acid cluster.

As described in greater detail herein, by exploiting the presentlydisclosed ability to identify the center position of a nucleic acidcluster, solid phase supports bearing high-density arrays of clusterscan be produced from which useful nucleic acid sequence information canbe obtained, where determination of the center position for adjacent,abutting or overlapping clusters, or for clusters from which overlappingor otherwise conflicting signals may emanate, permits unambiguousassignment of sequence information to its proper source cluster.

Accordingly and for example, the embodiments described herein identifythe center of a solid-phase nucleic acid cluster and therefrom willpermit the development of image processing software, as used to analyzeoptical signals that are generated during sequencing of such clusters,to discriminate unambiguously between two adjacent, abutting oroverlapping clusters in order to assign a sequencing signal to a single,discrete source cluster. These and related embodiments thus permitretrieval of meaningful information, such as sequence data, from regionsof high-density cluster arrays where useful information could notpreviously be obtained from such regions due to confounding effects ofoverlapping or very closely spaced adjacent clusters, including theeffects of overlapping signals (e.g., as used in nucleic acidsequencing) emanating therefrom.

As described in greater detail below, in certain embodiments there isprovided a composition that comprises a solid support having immobilizedthereto one or a plurality of nucleic acid clusters as provided herein.Each cluster comprises a plurality of immobilized nucleic acids of thesame sequence and has an identifiable center having a detectable centerlabel as provided herein, by which the identifiable center isdistinguishable from immobilized nucleic acids in a surrounding regionin the cluster. Also described herein are methods for making and usingsuch clusters that have identifiable centers.

The presently disclosed embodiments will find uses in numeroussituations where advantages are obtained from the ability to identify,determine, annotate, record or otherwise assign the position of asubstantially central location within a cluster, such as high-throughputnucleic acid sequencing, development of image analysis algorithms forassigning optical or other signals to discrete source clusters, andother applications where recognition of the center of an immobilizednucleic acid cluster is desirable and beneficial.

Nucleic Acids

In certain embodiments, the present invention contemplates methods thatrelate to high-throughput nucleic acid analysis such as nucleic acidsequence determination (e.g., “sequencing”). Exemplary high-throughputnucleic acid analyses include without limitation de novo sequencing,re-sequencing, whole genome sequencing, gene expression analysis, geneexpression monitoring, epigenetic analysis, genome methylation analysis,allele specific primer extension (APSE), genetic diversity profiling,whole genome polymorphism discovery and analysis, single nucleotidepolymorphism analysis, hybridization based sequence determinationmethods, and the like. One skilled in the art will appreciate that avariety of different nucleic acids can be analyzed using the methods andcompositions of the present invention.

The terms “nucleic acid”, “nucleic acid molecule”, and “polynucleotide”are used essentially interchangeably herein. In various embodiments,nucleic acids may be used as templates as provided herein (e.g., anucleic acid template, or a nucleic acid complement that iscomplementary to a nucleic acid nucleic acid template) for particulartypes of nucleic acid analysis, including but not limited to nucleicacid amplification, nucleic acid expression analysis, and/or nucleicacid sequence determination or suitable combinations thereof. Nucleicacids in certain embodiments include, for instance, linear polymers ofdeoxyribonucleotides in 3′-5′ phosphodiester or other linkages, such asdeoxyribonucleic acids (DNA), for example, single- and double-strandedDNA, genomic DNA, copy DNA or complementary DNA (cDNA), recombinant DNA,or any form of synthetic or modified DNA. In other embodiments, nucleicacids include for instance, linear polymers of ribonucleotides in 3′-5′phosphodiester or other linkages such as ribonucleic acids (RNA), forexample, single- and double-stranded RNA, messenger (mRNA), copy RNA orcomplementary RNA (cRNA), alternatively spliced mRNA, ribosomal RNA,small nucleolar RNA (snoRNA), microRNAs (miRNA), small interfering RNAs(sRNA), piwi RNAs (piRNA), or any form of synthetic or modified RNA.Nucleic acids used in the compositions and methods of the presentinvention may vary in length and may be intact or full-length moleculesor fragments or smaller parts of larger nucleic acid molecules. Inparticular embodiments, a nucleic acid may have one or more detectablelabels, as described elsewhere herein.

In particular embodiments, the nucleic acid to be used as a template orto be amplified, sequenced, analyzed, or otherwise used in a method setforth herein, can be at least 10, at least 20, at least 30, at least 40,at least 50, at least 50, at least 100, at least 150, at least 200, atleast 250, at least 500, or at least 1000 nucleotides in length. Inother particular embodiments, the nucleic acid can be about 150 to about4000 nucleotides in length, about 500 to about 3000 nucleotides inlength, or about 1000 to about 2000 nucleotides in length. Alternativelyor additionally, a nucleic acid can be at most 100, at most 250, at most500, at most 1000, at most 5000, at most 10,000, or at most 100,000nucleotides in length.

The terms “isolated nucleic acid”, “isolated polynucleotide”, and“isolated nucleic acid molecule” are used interchangeably herein andrefer to nucleic acid that is substantially or essentially free fromcomponents that normally accompany it in its native state, e.g.,polypeptides, cells, organisms, etc. In certain embodiments an “isolatednucleic acid” may be a nucleic acid that has been purified from thenucleotide sequences which flank it in a naturally-occurring state,e.g., a DNA fragment that has been separated or removed from thesequences that are normally adjacent to the fragment. In anotherembodiment, an “isolated nucleic acid” may refer to a nucleic acid thathas been extracted from, or separated or otherwise obtained from a cell,tissue, or organism such that it is no longer present in the cell,tissue or organism in its natural state.

It will be appreciated that nucleic acids of the present invention maybe obtained from any biological sample from a subject or biologicalsource. Biological samples may therefore include a blood sample, biopsyspecimen, tissue explant, organ culture, biological fluid or any othertissue or cell preparation, or fraction or derivative thereof orisolated therefrom, from a subject or a biological source. The subjector biological source may be a human or non-human animal, includingmammals and non-mammals, vertebrates and invertebrates, and may also beany other multicellular organism or single-celled organism such as aeukaryotic (including plants and algae) or prokaryotic organism,archaeon, microorganisms (e.g. bacteria, archaea, fungi, protists,viruses), aquatic plankton, a primary cell culture or culture adaptedcell line including but not limited to genetically engineered cell linesthat may contain chromosomally integrated or episomal recombinantnucleic acid sequences, immortalized or immortalizable cell lines,somatic cell hybrid cell lines, differentiated or differentiatable celllines, transformed cell lines, stem cells, germ cells (e.g. sperm,oocytes), transformed cell lines and the like. For example, nucleicacids may be obtained from primary cells, cell lines, freshly isolatedcells or tissues, frozen cells or tissues, paraffin embedded cells ortissues, fixed cells or tissues, and/or laser dissected cells ortissues. In certain embodiments, the nucleic acids may be derived,purified, or isolated from any known prokaryotic or eukaryotic organismor virus.

Exemplary prokaryotes include, but are not limited to, Escherichia coli,and species of Salmonella, Enterobacter, Camplyobacter, Stapylococcus,Pseudomonas, and Listeria.

Exemplary eukaryotes include, but are not limited to, humans andnon-human primates such as baboons, gorillas, chimpanzees, rhesusmacaques and other non-human primates, and also include equine, bovine,sheep (ovine), goat (caprine), pig (porcine), dog (canine), cat(feline), chicken, rat, and mouse (murine) species; other non-limitingexamples of eukaryotes include Xenopus laevis, Danio rerio, Drosophilamelanogaster, Caenorhabditis elegans, and yeast species (e.g.,Saccharomyces, Schizosaccharomyces, Clamydia). Other exemplaryeukaryotes include plants such as Arabidopsis thaliana, barley, citrusfruits, cotton, grapes, wheat, tomato, potato, sugar cane, maize,tobacco, poplars, rice, and soybean.

Exemplary viruses include, but are not limited to, adenoviruses,herpesviruses, poxviruses, parvoviruses, reoviruses, picornaviruses,togaviruses, orthomyxoviruses, rhabdoviruses, retroviruses (e.g.,lentiviruses), and hepadnaviruses. Other exemplary viruses include humanimmunodeficiency viruses and influenza viruses.

Nucleotides and Nucleosides

The term “nucleotide” refers to a heterocyclic nitrogenous base inN-glycosidic linkage with a phosphorylated sugar. Nucleotides areunderstood to include natural bases (standard), and a wide variety ofart-recognized modified bases. Such bases are generally located at the1″ position of a nucleotide sugar moiety. Nucleotides generally comprisea base, sugar and a phosphate group. In RNA, the sugar is a ribose, andin DNA the sugar is a deoxyribose, i.e., a sugar lacking a hydroxylgroup that is present in ribose. The nitrogenous base is a derivative ofa purine or a pyrimidine. The purines are adenosine (A) and guanidine(G), and the pyrimidines are cytidine (C) and thymidine (T) (or in thecontext of RNA, uracil (U)). The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine. Nucleotides are usually mono,di- or triphosphates. The nucleotides can be unmodified or modified atthe sugar, phosphate and/or base moiety, (also referred tointerchangeably as nucleotide analogs, nucleotide derivatives, modifiednucleotides, non-natural nucleotides, and non-standard nucleotides; seefor example, WO 92/07065 and WO 93/15187). Examples of modified nucleicacid bases are summarized by Limbach et al., (1994, Nucleic Acids Res.22, 2183-2196).

A nucleotide may also be regarded as a phosphate ester of a nucleoside,with esterification occurring on the hydroxyl group attached to C-5 ofthe sugar. The term “nucleoside” refers to a heterocyclic nitrogenousbase in N-glycosidic linkage with a sugar. Nucleosides are recognized inthe art to include natural bases (standard), and also to include wellknown modified bases. Such bases are generally located at the 1″position of a nucleoside sugar moiety. Nucleosides generally comprise abase and sugar group. The nucleosides can be unmodified or modified atthe sugar, and/or base moiety, (also referred to interchangeably asnucleoside analogs, nucleoside derivatives, modified nucleosides,non-natural nucleosides, or non-standard nucleosides). As also notedabove, examples of modified nucleic acid bases are summarized by Limbachet al., (1994, Nucleic Acids Res. 22, 2183-2196).

The terms “modified base” and “modified nucleotide” are usedinterchangeably herein and refer to nucleotide bases other than adenine,guanine, cytosine, thymine, and uracil at the 1′ position or theirequivalents; such bases can be used at any position, for example, withinthe catalytic core of an enzymatic nucleic acid molecule and/or in thesubstrate-binding regions of the nucleic acid molecule. The nucleosideresidues of a nucleic acid may be coupled to each other by any of thenumerous known internucleoside linkages. Such internucleoside linkagesinclude, for example and without limitation, phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone internucleotide linkages. Nucleic acidsmay contain a non-natural sugar moiety in the backbone. Exemplary sugarmodifications include but are not limited to 2′ modifications such asaddition of halogen, alkyl, substituted alkyl, allyl, aryl, O-alkyl orO-aryl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂,N₃, NH₂, heterocycloallyl, heterocycloallcaryl, aminoallylamino,polyallylamino, phosphate, substituted phosphate, substituted silyl, andthe like. Similar modifications can also be made at other positions onthe sugar, particularly the 3′ position of the sugar on the 3′ terminalnucleotide and the 5′ position of the 5′ terminal nucleotide. In certainembodiments, oligonucleotides may comprise polymers such as peptidenucleic acids (PNA) and locked nucleic acids (LNA).

Exemplary chemically modified and other natural nucleic acid bases thatcan be introduced into nucleic acids include, but are not limited to,inosine, dihydrouridine, hypoxathanine, isocytosine, isoguanine,N6-methyladenosine, pseudouracil, pyridin-2-one, pyridin-4-one,quesosine, threonine derivatives, uridine-5-oxyacetic acid, wybutosine,wybutoxosine, xathanine, β-D-galactosylqueosine, β-D-mannosylqueosine,2,2-dimethylguanosine, 15-halocytosine, 15-halouracil,1-methyladenosine, 1-methylinosine, 2-aminoadenine, 2-methyladenosine,2-methylguanosine, 2-methylthio-N-6-isopentenyladenosine, 2-propyladenine, 2-propyl guanine, 2-thiocytidine, 2-thiocytosine,2-thiothymine, 2-thiouracil, 2-thiouridine, 3-deazaadenine or the like,3-deazaguanine, 3-methyl uracil, 3-methylcytidine, 3-nitropyrrole,4-acetyltidine, 4-thiouracil, 4-thiouridine,5-(carboxyhydroxymethyl)uridine,5-carboxymethylaminomethyl-2-thiouridine, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g., ribothymidine),5-carboxymethylaminomethyluridine, 5-halo substituted uracil orcytosine, 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or6-alkylpyrimidines (e.g., 6-methyluridine), 5-hydroxymethyl cytosine,5-methoxyaminomethyl-2-thiouridine, 5-methyl-2-thiouridine,5-methylaminomethyluridine, 5-methylcarbonylmethyluridine,5-methylcytosine, 5-methyloxyuridine, 5-nitroindole, 5-propynylcytosine, 5-propynyl uracil, 5-uracil, 6-azo cytosine, 6-azo thymine,6-azo uracil, 6-methyl adenine, 6-methyl guanine, 7-deazaadenine,7-deazaguanine, 7-methyladenine, 7-methylguanine, 7-methylguanosine,8-amino adenine or guanine, 8-azaadenine, 8-azaguanine, 8-halo adenineor guanine, 8-hydroxyl adenine or guanine, 8-thioalkyl adenine orguanine, 8-thiol adenine or guanine or the like. In a particularembodiment, isocytosine and/or isoguanine may be used in a nucleic acidin order to reduce non-specific hybridization, as generally described inU.S. Pat. No. 5,681,702. Further examples of chemically modifiednucleotides that can be used in the methods and compositions of thepresent invention are those set forth below in the context of sequencingmethods.

Template Nucleic Acid

As used herein, the terms “template nucleic acid” and “nucleic acidtemplate” are used interchangeably and refer to the nucleic acid to beamplified, copied, sequenced, and/or otherwise analyzed. In certainembodiments the nucleic acid template may be in a single-stranded form,and in other embodiments, the nucleic acid template to be amplified,copied, sequenced, and/or otherwise analyzed may be provided in a doublestranded form. In certain embodiments, the template nucleic acidcomprises a target nucleic acid, and in certain embodiments the templatenucleic acid may comprise a target nucleic acid and in addition, one ormore non-target nucleic acid sequences. In related embodiments, thenucleic acid to be amplified, copied, sequenced, and/or otherwiseanalyzed comprises the target nucleic acid in single or double strandedform.

In particular embodiments, the template nucleic acid may be at least 10,at least 20, at least 30, at least 40, at least 50, at least 50, atleast 100, at least 150, at least 200, at least 250, at least 500, or atleast 1000 nucleotides in length. In other particular embodiments, thenucleic acid template may be about 150 to about 4000 nucleotides inlength, about 500 to about 3000 nucleotides in length, or about 1000 toabout 2000 nucleotides in length. Certain embodiments contemplate anucleic acid template of at most 100, at most 250, at most 500, at most1000, at most 5000, at most 10,000, or at most 100,000 nucleotides inlength.

Nucleic acid templates may be prepared using a variety of standardtechniques available and known to those skilled in the art. Exemplarymethods of template preparation include, but are not limited to, thosedescribed in U.S. Pat. No. 7,115,400 and U.S. Patent ApplicationPublication numbers 2005/0100900, 2005/0059048, 2007/0110638 and2007/0128624, each of which is herein incorporated by reference in itsentirety. The nucleotides making up the nucleic acid templates may benaturally occurring or non-naturally occurring nucleotides. Inparticular embodiments, a nucleic acid template may comprise one or moredetectable labels, as described elsewhere herein. The one or moredetectable labels may be attached to the nucleic acid template at the 5′end, at the 3′ end, and/or at any nucleotide position within the nucleicacid template. According to certain contemplated embodiments the nucleicacid templates of the invention not only may comprise the nucleic acidto be amplified and/or sequenced but may, in addition, optionallycomprise short oligonucleotide sequences at the 5′ and/or 3′ end(s) ofthe template nucleic acid.

The term “oligonucleotide” refers to a polymer of two or moredeoxyribonucleotides, ribonucleotides and/or naturally occurring orsynthetic analogs and derivatives thereof. Persons familiar with the artwill appreciate that any nucleic acid of the present invention mayinclude engineered internucleoside linkages, modified sugars, and/orPNA/LNA.

In certain embodiments, oligonucleotide sequences may be adapters thatare ligated to the 5′ and/or 3′ ends of a nucleic acid. In other certainembodiments, oligonucleotide sequences may already be present in thenucleic acid to be used as a template. In certain particularembodiments, adapters or other olignucleotide sequences may comprise oneor more detectable labels, as described elsewhere herein. The one ormore detectable labels can be attached at the 5′ end, at the 3′ end,and/or at any nucleotide position within the adapter or oligonucleotidesequence.

For example, the oligonucleotide sequence located at the 5′ end of anucleic acid template in certain embodiments may be referred to as “Y”.Oligonucleotide sequence Y can be of a known or unknown sequence or asuitable combination thereof and can be of variable length. Inparticular illustrative embodiments, for example, oligonucleotidesequence Y is 5, 10, 15, 20, 25, 30 or more nucleotides in length. Inother embodiments, oligonucleotide sequence Y is between about 5 andabout 100 nucleotides in length, between about 10 and about 50nucleotides in length, between about 10 and about 30 nucleotides inlength, or between about 5 and about 25 nucleotides in length, or anyintervening range lengths thereof. In certain embodiments,oligonucleotide sequence Y is approximately 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleotides in length. Naturally occurring and/ornon-naturally occurring nucleotides may be present in theoligonucleotide sequence Y.

In one embodiment, the oligonucleotide sequence located at the 3′ end ofa nucleic acid template may be referred to as “Z”. Oligonucleotidesequence Z can be of a known or unknown sequence or a suitablecombination thereof and can be of variable length. For example, inparticular illustrative embodiments, oligonucleotide sequence Z is 5,10, 15, 20, 25, 30 or more nucleotides in length. In other embodiments,oligonucleotide sequence Z is between about 5 and about 100 nucleotidesin length, between about 10 and about 50 nucleotides in length, betweenabout 10 and about 30 nucleotides in length, or between about 5 andabout 25 nucleotides in length, or any intervening range lengthsthereof. In certain embodiments, oligonucleotide sequence Z isapproximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotidesin length. Naturally occurring and/or non-naturally occurringnucleotides may be present in the oligonucleotide sequence Z.

The oligonucleotide sequences Y and Z located at the 5′ and 3′ ends,respectively, of a nucleic acid template need not be localized at theextreme ends of the template. In one embodiment, the oligonucleotidesequences Y and/or Z may be located at or near the 5′ and 3′ ends of anucleic acid template, respectively. For example, oligonucleotidesequences Y and/or Z may be within 0 to 100 nucleotides, within 5 to 50nucleotides, or within 10 to 25 nucleotides of the 5′ and 3′ ends. Incertain embodiments, oligonucleotide sequences Y and/or Z may be locatedmore than 50, more than 75, or more than 100 or more nucleotides awayfrom the 5′ and 3′ termini of the nucleic acid template. It will beappreciated that the oligonucleotide sequences Y and Z may therefore belocated at any position within the nucleic acid template. The sequencesY and Z are typically on opposing sides, i.e., flanking, a nucleic acidsequence which is to be amplified and/or sequenced. The skilled artisanwould also recognize that sequences Y and Z need not be equidistant fromthe 5′ and 3′ termini of the nucleic acid template, respectively.Adapters and oligonucleotide sequences, such as the oligonucleotidesequences Y and Z exemplified above, can be universal sequences.Universal sequences and their use for forming priming sites foruniversal primers are set forth in further detail below.

In particular embodiments, wherein the nucleic acid template is in adouble stranded form, the oligonucleotide sequences Y and Z arecontained at the 5′ and 3′ ends, respectively, of one of the strands.The other strand, due to the base pairing rules of DNA, is complementaryto the strand containing oligonucleotide sequences Y and Z and thus,contains an oligonucleotide sequence Z′ at the 5′ end and anoligonucleotide sequence Y′ at the 3′ end.

Nucleic Acid Complement

As used herein, the terms “complementary” and “complementarity” refer tonucleic acids (i.e., a sequence of nucleotides) that are related byWatson-Crick base-pairing rules according to convention in the molecularbiology art. Two nucleotides that are on opposite (i.e., with respect to573′ sugar-phosphate backbone polarity) complementary nucleic acidstrands and that are connected to one another via hydrogen bonds arecalled a base pair (often abbreviated bp). In the canonical Watson-Crickbase pairing, adenine (A) forms a base pair with thymine (T), as doesguanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced byuracil (U). For example, the nucleotide adenine is complementary tothymine. For example, the DNA sequence “A-G-T,” is complementary to theDNA sequence “T-C-A” or the RNA sequence “U-C-A”. Complementarity can be“partial,” in which only some of the nucleic acids' bases are pairedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands.

“Complementary nucleic acid”, “nucleic acid complement”, and “centralnucleic acid complement” are used substantially interchangeably andrefer to a nucleic acid complement or complementary nucleic acid that iscomplementary to a reference nucleic acid, such as a template nucleicacid. In certain embodiments, a nucleic acid complement may be generatedby “primer extension”, e.g., extending an oligonucleotide primer bypolynucleotide polymerase-catalyzed serial incorporation, proceeding ina 5′-to-3′ direction, into the thus-extended primer of individualnucleotides that are complementary to those of an antiparallel (e.g.,3′-to-5′) template nucleic acid sequence, wherein the primer is attachedat its 5′ end to a solid support and hybridized to the nucleic acidtemplate. In related embodiments, nucleic acid complements thus may beimmobilized to a solid support at their 5′ ends. In certain furtherrelated embodiments, copies of the immobilized nucleic acidcomplement(s) may be generated by performing nucleic acid amplification(e.g., primer extension, thermal polymerase chain reaction, isothermalpolymerase chain reaction, or other nucleic acid amplificationmethodologies). The nucleic acid complement may comprise any combinationof natural and/or non-natural nucleotides, e.g., nucleotide derivativesor analogs.

As also described elsewhere herein, an immobilized nucleic acidcomplement may advantageously comprise one or more detectable labels.The one or more detectable labels may be attached at the 5′ end, at the3′ end, and/or at any nucleotide position within the immobilized nucleicacid complement. For example, an immobilized primer may be extended, byprimer extension along a nucleic acid template, with a first nucleotidethat comprises one or more detectable labels, thereby generating adetectably labeled nucleic acid complement. In a related embodiment, theimmobilized primer may be extended by a primer extension reaction inwhich is included a mixture of nucleotides, wherein one or more of thenucleotides comprises one or more labels, thereby generating adetectably labeled nucleic acid complement.

In one non-limiting example, for the generation of an immobilizednucleic acid complement, an immobilized primer may be extended by aprimer extension reaction using a nucleotide mix comprising unlabeleddATP, dTTP, and dGTP and detectably labeled dCTP. The mixture may alsocomprise both labeled dCTP and unlabeled dCTP including, for example, asa mixture having a lesser amount of unlabeled dCTP relative to theamount of labeled dCTP. For instance, the amount of unlabeled dCTP maybe present in 1/10,000, 1/1,000, 1/100, 1/20, 1/10, ⅕, ⅓, or ½ therelative amount of the labeled dCTP in the primer extension reaction.Alternatively, multiple labeled nucleotides may be present in the primerextension reaction for incorporation into the nucleic acid complement.For example, in the generation of an immobilized nucleic acidcomplement, a primer may be extended using a nucleotide mix comprisingunlabeled dTTP and dGTP and detectably labeled dATP and dCTP. As anotherexample, any one, two, three, or all four of dATP, dTTP, dGTP and dCTPmay be detectably labeled and incorporated into the extended primer toeffect the generation of a detectably labeled immobilized nucleic acidcomplement. Different labeled nucleotides used in a mixture, such asthose exemplified above, can have the same label or, alternatively,different labels can be present on the different labeled nucleotides.

Nucleic Acid Clusters

“Nucleic acid cluster” and “nucleic acid colony” are usedinterchangeably and refer to a plurality of copies of a nucleic acidtemplate and/or complements thereof attached to a solid support.Typically and in certain preferred embodiments, the nucleic acid clustercomprises a plurality of copies of template nucleic acid and/orcomplements thereof, attached via their 5′ termini to the solid support.The copies of nucleic acid strands making up the nucleic acid clustersmay be in a single or double stranded form. Copies of a nucleic acidtemplate that are present in a cluster can have nucleotides atcorresponding positions that differ from each other, for example, due topresence of a label moiety. The corresponding positions can also containanalog structures having different chemical structure but similarWatson-Crick base-pairing properties, such as is the case for uracil andthymine.

The nucleic acid clusters of the invention can have different shapes,sizes and densities depending on the conditions used. For example,clusters can have a shape that is substantially round, multi-sided,donut-shaped or ring-shaped. The diameter of a nucleic acid cluster canbe designed to be from about 0.2 μm to about 6 μm, about 0.3 μm to about4 μm, about 0.4 μm to about 3 μm, about 0.5 μm to about 2 μm, about 0.75μm to about 1.5 μm, or any intervening diameter. In a particularembodiment, the diameter of a nucleic acid cluster is about 0.5 μm,about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 4μm, about 5 μm, or about 6 μm. The diameter of a nucleic acid clustermay be influenced by a number of parameters, including, but not limitedto the number of amplification cycles performed in producing thecluster, the length of the nucleic acid template or the density ofprimers attached to the surface upon which clusters are formed. Thedensity of nucleic acid clusters can be designed to typically be in therange of 0.1/mm², 1/mm², 10/mm², 100/mm², 1,000/mm², 10,000/mm² to100,000/mm². The present invention further contemplates, in part, higherdensity nucleic acid clusters, for example, 100,000/mm² to 1,000,000/mm²and 1,000,000/mm² to 10,000,000/mm².

As disclosed herein, a nucleic acid cluster may comprise (a) asubstantially central location as provided herein, comprising at leastone nucleic acid template and at least one nucleic acid complementthereof, and (b) a surrounding region comprising one or more immobilizedcopies of the at least one nucleic acid template and of the at least onenucleic acid complement thereof, the surrounding region comprising thatportion of the cluster that is outside of the substantially centrallocation. In certain preferred embodiments, within the substantiallycentral location either or both of the nucleic acid template and thenucleic acid complement thereof comprise a detectable label, such thatthe nucleic acid cluster has an identifiable center as also discussedelsewhere herein.

Accordingly, in certain preferred embodiments, there is contemplated,immobilized on a solid support as provided herein, one or a plurality ofnucleic acid clusters that each have an identifiable center. Withoutwishing to be bound by any particular theory, it is believed thatnucleic acid clusters are generated by the geometric and radial nucleicacid amplification of a single immobilized nucleic acid template and/ora single immobilized nucleic acid complement of the template (i.e., theoriginal nucleic acid template and the original complement thereof).Thus, detectably labeling the immobilized nucleic acid template and/orthe initial nucleic acid complement permits ready identification of thecenter of a nucleic acid cluster generated therefrom.

Identifiable Center of a Nucleic Acid Cluster

The identifiable center of a nucleic acid cluster comprises one or morenucleic acids in a cluster that can be distinguished from surroundingnucleic acids in the same cluster. Typically, the one or more nucleicacids in the identifiable center and the surrounding nucleic acids sharea common sequence, for example, by being copies of a common templatenucleic acid or of a nucleic acid complement of the template nucleicacid. In particular embodiments, the nucleic acids in the center regionof a cluster may include nucleotide analogs that are different from thenucleotide analogs found in nucleic acids present in the surroundingregion. For example, nucleic strands in the center region of a clustercan include uracil nucleotides at one or more position in a sequencewhereas strands in the surrounding region can have thymine nucleotidesat the one or more positions in the sequence. Other analog pairs such asguanine and 8-oxo guanine find similar use.

The identifiable center of a nucleic acid cluster may in certainembodiments comprise the original template nucleic acid and the originalimmobilized complement thereof, either of which, or both, can bedetectably labeled. In other embodiments, the identifiable center of anucleic acid cluster may comprise a detectably labeled nucleic acidcomplement and/or a detectably labeled template nucleic acid and/ordetectably labeled copies thereof.

The identifiable center of a nucleic acid cluster may comprise adetectably labeled nucleic acid complement of a nucleic acid templateand/or detectably labeled copies thereof. In certain embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more copies of the nucleic acid complementmay be detectably labeled with a detectable label as provided herein. Incertain other embodiments, less than 25, less than 20, less than 15,less than 10 or less than 5 copies of the nucleic acid complement may bedetectably labeled. Additionally or alternatively, the identifiablecenter of a nucleic acid cluster may in certain embodiments comprise adetectably labeled nucleic acid template and/or detectably labeledcopies thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore copies of the nucleic acid template may be detectably labeled. Inother certain embodiments, less than 25, less than 20, less than 15,less than 10 or less than 5 copies of the nucleic acid template arelabeled.

The identifiable center of a nucleic acid cluster may be identified bydetecting a detectably labeled nucleic acid complement and/or adetectably labeled template nucleic acid and/or detectably labeledcopies thereof, using a suitable imaging means, for example, a confocalimaging device or a charge coupled device (CCD) camera. Exemplaryimaging devices, include, but are not limited to those described in U.S.Pat. No. 7,329,860; U.S. Pat. No. 5,754,291; U.S. Pat. No. 5,981,956 andU.S. Ser. No. 12/295,337, each of which is herein incorporated byreference in its entirety.

It will be appreciated that the imaging means may be used to determine areference position in a nucleic acid cluster or in a plurality ofnucleic acid clusters on the solid support, such as the location,boundary, diameter, area, shape, overlap and/or center of one or aplurality of solid-phase immobilized nucleic acid clusters (and/or of adetectable signal originating therefrom), and that such referenceposition may be recorded, documented, annotated, converted into aninterpretable signal, or the like, as may appropriately yield meaningfulinformation depending on the methodologies and instrumentation beingemployed. For example, the reference position can be interpreted by theimaging device as a signal that may be generated from a labeled centerin a cluster, from a surrounding region within the cluster, and/or fromtwo or more adjacent, abutting or overlapping clusters. The signal may,for instance, take the form of a detectable optical signal emanatingfrom a defined and identifiable location, such as a fluorescent signal,or may be a detectable signal originating from any other detectablelabel as provided herein.

Certain embodiments contemplate determination of a reference position byidentification of physical locations on the solid support that lack adetectable label, such as (in certain embodiments) the surroundingregion that surrounds the substantially central location in animmobilized nucleic acid cluster. The reference position thus mayidentify the actual physical position of the substantially centrallocation within the cluster, or of the surrounding region within thecluster which surrounding region surrounds the substantially centrallocation in the cluster, or of two or more overlapping clusters that areimmobilized on the solid support, depending on the particular formatthat is being employed according to the disclosure herein.

According to these and related embodiments as will be apparent to theskilled person in view of the present disclosure, the referencedposition of a signal generated from a substantially central location ofa cluster, and/or from another location in an immobilized nucleic acidcluster, and/or from two or more overlapping clusters, may be used todetermine the actual physical position on the solid support of thecenter, and/or of the cluster, and/or of two or more adjacent, abuttingor overlapping clusters, which may be understood typically to includeimmediately proximate or adjacent clusters from which signals emanate ina manner that precludes ready identification of the single sourcecluster for a given signal, e.g., overlapping signals.

The position of the identifiable center may be determined and recordedprior to, during or after, amplification of the detectably labelednucleic acid complement and/or of the detectably labeled templatenucleic acid, which amplification takes place as a step in the presentlydescribed methods for producing a nucleic acid cluster having anidentifiable center, including in certain embodiments methods whichfurther comprise performing one or more nucleic acid sequencing steps.In yet other embodiments, the position of the identifiable center (i.e.,the origin of the cluster as identified by detection of the detectablelabel that may be a component of the nucleic acid template or of theimmobilized nucleic acid complement of such template, or that may be acomponent of both) is determined and recorded before any nucleic acidamplification step takes place, and optionally also after each of one ora plurality of amplification reactions that amplify the nucleic acidcomplement, the nucleic acid template, and/or copies thereof. In thismanner, the center of the nucleic acid cluster, comprising at least onedetectably labeled nucleic acid, can be distinguished from surroundingcopies that are not labeled, or that may have a different detectablelabel.

Alternatively, the substantially central location in a cluster (e.g.,the site where reside the nucleic acid template and the nucleic acidcomplement thereof, at least one of which is detectably labeled) may bedistinguished due to the absence therefrom of a signal for a distinctlabel that is present only in the surrounding nucleic acid copies.Absence of a signal for a label can be due to the absence of the labelfrom the location being detected or absence of signal can be due to anagent that quenches or masks the label, thereby preventing or inhibitingdetection of the label. In related further embodiments, the position ofthe identifiable center may be determined and recorded beforecommencing, or during each of one or a plurality of sequencing steps asprovided herein, which sequencing steps are present in a method thatcomprises determining the nucleotide sequence of the nucleic acidtemplate and/or of the nucleic acid complement thereof in an immobilizednucleic acid cluster.

In an exemplary embodiment, the identifiable center of a cluster may bemade by carrying out the first cycles of template amplification using anucleoside analog which is not used in the later amplification cycles.As such, the nucleoside analog is incorporated into nucleic acid strandsin the center region of the cluster but not in nucleic acid strandswhich form the surrounding region. For example, nucleosides such asuracil or 8-oxo-guanine can be used to selectively form strands in thecentral location of the clusters that contain uracil or 8-oxo-guaninebases respectively, while the strands in the surrounding region lackuracil or 8-oxo-guanine. The clusters can be treated to selectivelyremove the nucleic acids in the central location while leaving those inthe surrounding region. Continuing with the example of a cluster havinguracil or 8-oxo-guanine bases in the nucleic acids of the centrallocation, the clusters can be treated to remove the uracil or8-oxo-guanine bases from their sugars. This will give rise to strands inthe central location that have abasic sites which undergo strandcleavage in the presence of endonucleases while strands in thesurrounding region are not cleaved. The resulting cluster will have aring or donut shape.

FIG. 3 shows an image of an array of clusters where the central locationof each cluster appears darker than the surrounding region. The clusterswere produced using dUTP plus dATP, dGTP and dCTP in the first fewcycles of amplification, followed by dTTP, dATP, dGTP and dCTP in theremaining cycles. After cluster formation, the solid support was treatedwith USER (New England Biolabs, Ipswich, Mass.; product number M5505).The USER enzyme generates a single nucleotide gap at the location of auracil. USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and theDNA glycosylase-lyase Endonuclease VIII. UDG catalyzes the excision of auracil base, forming an abasic (apyrimidinic) site while leaving thephosphodiester backbone intact. The lyase activity of Endonuclease VIIIbreaks the phosphodiester backbone at the 3′ and 5′ sides of the abasicsite so that base-free deoxyribose is released. After USER treatment,the strands are thus modified such that they are not capable of beinglabeled by an intercalator stain (in this case Sybr Green). Similarly,the modified strands are not capable of undergoing hybridization with asequencing primer, so are not detectable upon sequencing. The dark spaceon the image of FIG. 3 provided by the central regions can provide afiducial marking for image registration or can provide a dark regionuseful for various focusing techniques.

The identifiable center of a nucleic acid cluster as provided by thecompositions and methods described herein will be understood by thoseskilled in the art to include, in non-limiting fashion, the approximategeometric center, the approximate center of mass, or the centroid of anucleic acid cluster. Thus, the approximate center would be understoodto be substantially the center location of the nucleic acid cluster. Asused herein, the terms “substantially center location”, “substantiallycentral location”, “substantially central position”, and “substantiallycenter position” are used interchangeably and refer to a center locationor position that is located at a site which comprises the at least 90%,92%, 95%, 97% or 99% confidence interval of the geometric center, centerof mass, or centroid of the nucleic acid cluster, as can be determinedusing available image analysis algorithms to assess statisticalsignificance Images of nucleic acid clusters can be analyzed using imageanalysis software, for example, the image analysis software asimplemented in the Illumina data analysis pipeline (Illumina, Inc., SanDiego, Calif.). A cluster will typically approximate a circle shape, butit will be understood that clusters of other shapes can be used as welland that the methods set forth herein can be used for clusters of any ofa variety of shapes.

The identifiable center, while being discrete, may also vary in size. Inparticular embodiments, the identifiable center comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, or more labeled nucleic acids, each of whichcomprises one or more detectable labels. For example, the area of theidentifiable center of a nucleic acid cluster can be between about1/100,000^(th) to about 1/1,000,000^(th) of the nucleic acid clusterarea, between about 1/10,000^(th) to about 1/100,000^(th) of the nucleicacid cluster area, between about 1/1,000^(th) to about 1/10,000^(th) ofthe nucleic acid cluster area, between about 1/100^(th) to about1/1,000^(th) of the nucleic acid cluster area, or between about1/10^(th) to about 1/100^(th) of the nucleic acid cluster area, or anyintervening range thereof. In particular embodiments, the area of theidentifiable center of a nucleic acid cluster can be at least about1/100,000^(th), 1/10,000^(th), 1/1,000^(th), 1/100^(th), 1/10^(th),⅕^(th), ⅓^(rd), or ½ of the nucleic acid cluster area. Alternatively oradditionally, the area of the identifiable center of a nucleic acidcluster can be at most about 1/1,000,000^(th), 1/100,000^(th),1/10,000^(th), 1/1,000^(th), 1/100^(th), 1/10^(th), ⅕^(th), ⅓^(rd), or ½of the nucleic acid cluster area. One having skill in the art wouldunderstand that the size of the identifiable center can be limited, inpart, by the number of nucleic acid amplification reactions performed onthe central nucleic acid complement and/or the nucleic acid template.

Detectable Labels

Conventional detectable labels are contemplated for use in certainherein described embodiments to permit determination of the location ofa nucleic acid such as a nucleic acid template and/or an immobilizedcomplement thereof, and a large number of such labels are known fornucleic acid labeling, typically comprising chemical or biochemicalmoieties having known physicochemical and/or biochemical properties.Persons skilled in the art will appreciate that a detectable label maybe a moiety the presence or absence of which can be ascertained withconfidence and in a statistically significant manner relative to asuitable control, using state of the art instrumentation andmethodologies.

Detection of a detectable label may be carried out by any suitablemethod as may be known according to the particular detectable label thatis employed, including, as non-limiting examples, by fluorescenceimaging or by other imaging means, such as by laser scanning confocalmicroscopy and/or by using a charge coupled device (CCD) camera. Imagingmeans suitable for determining fluorescent signals produced uponappropriate excitation by detectable labels that are fluorophores aredescribed, for example, in PCT application number PCT/US2007/07991,which is herein incorporated by reference in its entirety.

In particular embodiments, one or more detectable label may bechemically conjugated to nucleic acid such as a template or primer. Forexample, a detectable label can be present on a template nucleic acidprior to attachment of the template to a surface and/or prior toamplification of the template to form a cluster. If desired, a primerused for one or more amplification steps, for example, in the earlystages of cluster growth can include one or more detectable labels.Alternatively or additionally, nucleotides used during particularamplification steps, such as early steps of cluster growth, can containa detectable label. Thus, a detectable label may be chemicallyconjugated to a naturally occurring or artificial nucleotide or to anucleotide analog, a nucleotide derivative, or a modified or non-naturalnucleotide.

Detectably labeled nucleic acids, nucleotides, derivatives, or analogsthereof may comprise one or more detectable labels. Detectably labeledprimers or nucleotides may be incorporated into nucleic acids (e.g., byextension, polymerization, and/or ligation), to render the resultingnucleic acids detectable. Each strand of a nucleic acid generated byprimer extension or nucleic acid amplification (e.g., thermal polymerasechain reaction or isothermal polymerase chain reaction, or other nucleicacid amplification strategies) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more detectably labeled nucleotides. In certain embodiments, anucleic acid generated by primer extension or nucleic acid amplificationmay comprises a single detectably labeled nucleotide, and in certainembodiments the nucleic acid may comprise at least one, at least two ormore detectably labeled nucleotides. In other embodiments, a nucleicacid generated by primer extension or nucleic acid amplificationcomprises exclusively detectably labeled nucleotides. A nucleic acidgenerated by primer extension or nucleic acid amplification may comprisea single type of detectable labeled nucleotide such as, for example, alabeled form of A, G, C, T or U. In certain embodiments, 1, 2, 3, 4 orall of the nucleotides of a particular type may include a detectablelabel.

In particular embodiments, labeled nucleotides can be used exclusivelyin the initial cycles of extension in order to exclusively label thecenter location of a cluster. For example, labeled nucleotides may beused in the first and second cycles of extension, with subsequent cyclesof extension/amplification performed using solely unlabellednucleotides. For example, if the template strand being copied in theextension step contains 1000 nucleotide bases, 250 of each type ofnucleotide (A, G, C and T/U), the strand generated upon extension maycontain between 1 to 1000 labeled nucleotides. If the extension iscarried out with labeled A nucleotides, and unlabelled G, C and T/Unucleotides, the extension product should contain 250 labels, one perincorporated A nucleotide. Using a 1/1 ratio of labeled to unlabeled Ameans that the strands should contain 125 labels, assuming equivalentincorporation kinetics for the two A nucleotides. Thus the clusters maycontain a central labeled strand carrying multiple labels, surrounded byunlabelled strands.

In certain preferred embodiments the preferred detectable label is afluorophore. Exemplary fluorophores, include, but are not limited toumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,tetramethyl rhodamine (TMR), eosin, green fluorescent protein,erythrosin, coumarin, alexa, BODIPY, acridine, coumarin, benzanthracene,cyanins methyl coumarin, pyrene, malachite green, stilbene, luciferyellow, Cascade Blue™, Texas Red, dichlorotriazinylamine fluorescein,dansyl chloride, phycoerythrin, fluorescent lanthamide complexes such asthose including Europium and Terbium, Cy3, Cy5, molecular beacons andfluorescent derivatives thereof, as well as others known in the art asdescribed, for example, in Principles of Fluorescence Spectroscopy,Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999)and the 6th Edition of the Molecular Probes Handbook by Richard P.Haugland; Welch et al. (Chem. Eur. J. 5(3): 951-960, 1999); Zhu et al.(Cytometry 28:206-211, 1997); Prober et al. (Science 238:336-341, 1987);Connell et al. (BioTechniques 5(4):342-384, 1987); Ansorge et al. (Nucl.Acids Res. 15(11):4593-4602, 1987); and Smith et al. (Nature 321:674,1986). Other commercially available fluorescent labels include, but arenot limited to, FAM, HEX, TAMRA and Rox (see, e.g., Premstaller et al.,2002 Genomics 79(6):793). The fluorophores may be rhodamine or cyanineanalogues, for example as described in WO 07/135,368 and U.S. Ser. No.12/227,474. Example of commercially available fluorophores include thosesupplied by Atto-Tec GmbH (Siegen-Weidenau, Germany) or Dyomics GmbH(Jena, Germany). Fluorescent nanocrystals can be useful as well due totheir relatively narrow excitation and emission profiles, which can beconvenient for distinguishing from other dyes used in a particularmethod or technique.

Although fluorescent labels are preferred, other forms of detectablelabels will be apparent to those skilled in the art, as useful in theherein disclosed compositions and methods. For example, in particularembodiments, the detectable label may comprise one or more ofradionuclides, enzymes, chemiluminescent agents, and/or colorimetric orchromogenic agents. Nucleic acids and/or nucleotides comprising one ormore of these or other detectable labels as provided herein may bereferred to as being detectably labeled.

In other embodiments, detectable labels may include, but need not belimited to, radiolabels, quantum dot labels, detectable metal labels(e.g., gold, such as colloidal gold, silver), haptens (e.g.,dinitrophenol, digoxigenin, biotin), chromophores, enzymes, affinityligands, electromagnetic spin labels, heavy atom labels, nanoparticlelight scattering labels or other labeled nanoparticles or sphericalshells, and other signal generating labels known to those of skill inthe art (see, for example, Empodocles, et al., Nature 399:126-130, 1999;Reichert et al., Anal. Chem. 72:6025-6029, 2000; and Lacoste et al.,Proc. Natl. Acad. Sci. USA 97 (17):9461-9466, 2000. Affinity ligands canbe detected, for example, by binding to a receptor having a detectablelabel. For example, the SAPE technique can be used in which nucleicacids having biotin are incubated with streptavidin-phycoerythrin(SAPE), followed by incubation with a biotinylated anti-streptavidinantibody, and finally incubation with SAPE again. Exemplary SAPE methodsare described in U.S. Pat. No. 6,203,989, which is incorporated hereinby reference.

Affinity ligands and receptors, when present as labels on nucleic acidsaccording to the methods set forth herein, can be used to attach thenucleic acids to surfaces. For example, a template nucleic acid orcomplementary copy of a template that is chemically conjugated orotherwise attached to an affinity ligand or receptor can be attached toa surface via binding to a partner receptor or ligand on the surface.

Exemplary radiolabels include, but are not limited to ¹⁴C, ⁹⁰Y, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, Tc99m, ³⁵S and ³H.

Exemplary enzymes include, but are not limited to, readily detectablereporter enzymes, for example, enzymes having well characterized andreadily available substrates from which detectable products can begenerated, such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, and acetylcholinesterase.

Multiple detectable labels may also be used in certain contemplatedembodiments of the invention. For example, bi-fluorophore fluorescenceresonance energey transfer (FRET) cassettes (Tet. Letts. 46:8867-8871,2000) are well known in the art and may be utilized as detectable labelsas provided herein. Multi-fluor dendrimeric systems (J. Amer. Chem. Soc.123:8101-8108, 2001) may also be used.

Multi-component labels may also be used in certain of the hereindescribed invention embodiments. A multi-component label is one which isdependent on the interaction with a further compound for detection. Forexample, a common multi-component label used in biology is thebiotin-streptavidin system. Biotin may be used as a label attached tothe nucleotide base. Streptavidin may then be added separately to enabledetection of the location of the biotin to occur, which detection mayproceed, for example, by providing streptavidin in the form of afluorescent conjugate (e.g., fluorescein-streptavidin) or as an enzymeconjugate (e.g., horseradish peroxidase-streptavidin) or as a reagentthat comprises another detectable label as provided herein. SAPEmethods, such as those set forth above are another example ofbiotin-streptavidin systems. Other multi-component systems areavailable. For example, the chemical hapten dinitrophenol (DNP) has anumber of specific commercially available fluorescent antibodies thatmay be used for detection, by a variety of immunochemical detectionmeans, of detectably labeled molecules that comprise DNP as thedetectable label.

Detectable Center Labels

As used herein, the term “detectable center label” refers to adetectable label, as described supra, that may be detected to identify asubstantially central location of a nucleic acid cluster, for example,in the course of annotating the center position of the cluster.Preferably a detectable center label may be used to identify anidentifiable center of a nucleic acid cluster as provided herein, suchthat in certain embodiments the detectable center label may be limitedin its occurrence or distribution to a substantially central location ina cluster. A detectable center label may typically be selected so thatit can be readily distinguished from any other detectable label(s) thatmay be used to mark other than the identifiable center of a cluster,such as labels used in nucleic acid sequencing within a cluster and/orlabels situated in a surrounding region in a cluster, which surroundingregion surrounds the substantially central location of the cluster. Acenter label can be a different molecule than those used as labels insequencing. Alternatively, the label may be similar to, or identical toone or more of the labels used in sequencing. The label may be attachedto the central region of the cluster in such a way that the label can bequenched or removed, for example, by chemical cleavage. Thus an image ofthe central region of the clusters can be recorded and the label removedbefore commencing sequencing. In such a configuration, the need foradditional hardware on the sequencing system may be removed.

A detectable center label may be incorporated into the nucleotidesequence of the original nucleic acid template and/or the originalimmobilized nucleic acid complement thereof, so as to allow theidentification of the identifiable center of a nucleic acid cluster. Inparticular embodiments, one or more detectable center labels may beincorporated into one or a plurality of copies of the template nucleicacid and/or immobilized nucleic acid complement thereof. In certainembodiments, the detectable center label may be incorporated into 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50 or more copies of the templatenucleic acid and/or immobilized nucleic acid complement thereof. Incertain embodiments labeled nucleotides may be incorporated in the firstand second amplification cycles such that both primer 1 and primer 2 areextended using labeled nucleotides in each cluster. If primer 1 orprimer 2 contains a cleavage site for subsequent cluster linearization,use of labeled nucleotides in two or more cycles avoids loss of labeledstrand upon linearization which could otherwise result if only the firstextension cycle uses labeled nucleotides.

In embodiments where labeled nucleotides are incorporated, the label maybe attached to the nucleotide through a cleavable linker. Some usefulnucleotides with cleavable linkers are described in U.S. Pat. No.7,414,116 and U.S. Pat. No. 7,057,026, the contents of which areincorporated by reference herein in their entirety. Examples ofcleavable linkers include chemically cleavable linkers, photochemicallycleavable linkers or enzyme cleavable linkers. Chemically cleavablelinkers may include linkers containing an allyl, disulfide or azidomoiety. A linker may be cleaved after determining the central locationof a cluster, and the cleaving step can occur before or after a firstcycle of sequencing. In particular embodiments, a linker used toidentify a central location may be of the same moiety as a linker whichattaches a base to a detectable label of the nucleotides used insequencing. An advantage of using the same linker for identifying acentral location and for sequencing steps is that a single cleavagetreatment may remove both the sequencing label and the central labelfrom the clusters.

The detectable center label identifies a substantially central locationor center of the cluster. In certain embodiments, one or a plurality ofcopies of the template nucleic acid, and/or of the immobilized nucleicacid complement thereof, surround the substantially central location orcenter of the nucleic acid cluster and are not detectably labeled. Incertain related embodiments, the one or a plurality of copies of thetemplate nucleic acid, and/or of the immobilized nucleic acid complementthereof, surround the substantially central location or center of thenucleic acid cluster and comprise a detectable label that is detectablydistinct from (i.e., can be readily discerned from) the detectablecenter label. Two detectable labels that are detectably distinct may bereadily discerned from one another by methodologies with which theskilled person will be familiar.

For instance, if two detectable labels are both fluorophores, theskilled person can select two or more different fluorophores that aredetectably distinct by virtue of their having readily distinguishableexcitation/emission spectra, such that appropriate detection means(e.g., excitation energy sources, defined band-pass filters, sensorstuned to specific emissions wavelengths or wavelength ranges,photomultiplier tubes, etc.) can be selected to afford unambiguousdiscrimination between two discrete labels. Similarly, two or moredetectable labels may be selected having distinct detection means, suchas a first detectable label that is a fluorophore and a second (andoptionally third, fourth, etc.) detectable label that may be selectedfrom a visible-light chromophore, a radiolabel, and/or any other type ofdetectable label as known in the art or provided herein. Thus, accordingto these and related embodiments, the compositions and methods of thepresent invention provide, in pertinent part, a nucleic acid clusterhaving an identifiable center, wherein the center region in the clustercan be distinguished from the surrounding region in the cluster.

Various embodiments of the present invention also contemplate, in part,methods for determining the nucleotide sequence(s) of one or morenucleic acid clusters that each have a detectable center label, whereineach detectable center label is detectably distinct, i.e.,distinguishable, from any detectably labeled sequencing nucleotides thatmay be used in a sequencing reaction. In certain other embodiments,however, sequencing methods are contemplated in which a nucleic acidcluster to be sequenced may comprise a detectable center label that isnot detectably distinct, i.e., that may be indistinguishable from anydetectably labeled sequencing nucleotides that are used in a sequencingreaction to determine the nucleic acid sequence.

Oligonucleotide Primers

An oligonucleotide primer may be a nucleic acid molecule as providedherein that includes a sequence of contiguous nucleotides that iscapable of hybridizing to a complementary polynucleotide sequence andinitiating a polynucleotide polymerase reaction, including, but notlimited to a primer extension, a thermal polymerase chain reaction, anisothermal polymerase chain reaction, or certain otherpolymerase-catalyzed extension and/or amplification reactions.Oligonucleotide primers may be about 5, about 10, about 15, about 16,about 18, about 20, about 22, about 24, or about 25 nucleotides inlength, in certain embodiments. In other embodiments, oligonucleotideprimers may be between about 5 and about 100 nucleotides in length,between about 10 and about 50 nucleotides in length, between about 15and about 30 nucleotides in length, or between about 18 and about 25nucleotides in length, or any intervening range lengths thereof.Naturally occurring or non-naturally occurring nucleotides may bepresent in an oligonucleotide primer as contemplated herein. In certainembodiments, the oligonucleotide primers of the invention may compriseone or more detectable labels.

Certain preferred embodiments of the present invention contemplateoligonucleotide primers that are attached at their 5′ ends (or 5′termini) to a solid support. As used herein, the terms “colony primer”and “cluster primer” are used interchangeably and refer to a primer thatis attached at its 5′ end to a solid support, to provide an immobilizedoligonucleotide primer. The immobilized primer may serve as an extensionand/or amplification primer for the generation of an immobilized nucleicacid complement of a nucleic acid template, and/or for the generation ofone or a plurality of immobilized copies of the nucleic acid templateand/or of the immobilized nucleic acid complement. A plurality of one ortwo different colony primers may be used to generate nucleic acidcolonies in certain embodiments of the present invention.

In one non-limiting example, a plurality of nucleic acid templates thatcomprise the nucleic acid sequences to be amplified, wherein the nucleicacid templates contain at their 5′ ends an oligonucleotide sequence Yand at their 3′ ends an oligonucleotide sequence Z, are contacted with aplurality of colony primers X, which can hybridize to theoligonucleotide sequence Z and carry at the 5′ end a means for attachingthe oligonucleotide primers to a solid support. In such instancessequences Y and Z can function as universal sequences that are common toall members of the plurality of templates, and X can be a universalsequence which is capable of hybridizing to all members of the pluralityof templates.

In another non-limiting example, a plurality of oligonucleotide primerscomprising a first and a second immobilized oligonucleotide primer X arecontacted with the plurality of nucleic acid templates. Preferably,oligonucleotide sequence Z can hybridize to the first immobilizedoligonucleotide primer X and the oligonucleotide sequence Y includes asequence that is the same as the second immobilized oligonucleotideprimer. The amplification can be carried out with a single immobilizedprimer in embodiments wherein sequences Z and Y are complementary (i.e.,the ends of the templates are self-complementary).

Hybridization

Hybridization refers to the base pairing of a nucleic acid to itscomplement. The nucleic acid hybrids may be deoxyribonucleic acid (e.g.,DNA duplexes), ribonucleic acid (e.g., RNA duplexes), or a combinationthereof (e.g., DNA/RNA heteroduplexes). The specificity of singlestranded DNA to hybridize complementary fragments is determined by the“stringency” of the reaction conditions. Hybridization stringencyincreases as the propensity to form duplexes/heteroduplexes decreases.

One or more oligonucleotide primers (e.g., colony primers) may hybridizeto the 3′ end of a target nucleic acid template or a complementarynucleic acid thereof (e.g., a central nucleic acid complement). Incertain embodiments, the oligonucleotide primer may be 90%, 95%, 99% or100% complementary to (e.g., identical to the complement of) the targetnucleic acid. In one embodiment, the oligonucleotide primer may be 100%complementary (i.e., completely complementary or primer-specific) to thetarget nucleic acid sequence. In nucleic acid amplification andsequencing reactions, high stringency conditions are generally employedto favor specific primer/target hybridizations, which can be used tospecifically amplify or extend target nucleic acid sequences comprisingthe primer-specific binding sites.

It is well understood that the stability of nucleic acid duplexes isinfluenced by: (1) the number of complementary nucleotide base pairs,(2) the type of base pairs, (3) salt concentration (ionic strength) ofthe reaction mixture, (4) the temperature of the reaction, and (5) thepresence of certain organic solvents, such as formamide which decreasesnucleic acid duplex stability. In general, the longer theoligonucleotide, the higher the temperature required for properhybridization. A common approach is to vary the temperature: higherrelative temperatures result in more stringent reaction conditions.(Ausubel et al., 1987) provide an excellent explanation of stringency ofhybridization reactions.

To hybridize under “stringent conditions” describes hybridizationprotocols in which nucleotide sequences at least 60% identical to eachother remain hybridized. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium. Sincethe target sequences are generally present at excess, at Tm, 50% of theoligonucleotides are occupied at equilibrium.

Universal Primers

According to certain embodiments of the compositions and methods thatare described herein, universal primers are used to generate, amplify,sequence and/or otherwise analyze nucleic acid templates and theircomplements. The skilled person will recognize that universal primerscan amplify variable template nucleic acid sequences (e.g., “target”nucleic acids) that are flanked at their 5′ and 3′ ends by a common oruniversal sequence. A universal sequence is a region of nucleotidesequence that is common to, i.e., shared by, two or more nucleic acidmolecules, where the two or more nucleic acid molecules also haveregions of sequence differences. A universal sequence that may bepresent in different members of a collection of nucleic acid moleculescan allow the replication or amplification of multiple differentsequences using a single universal primer that is complementary to theuniversal sequence. Similarly, at least one, two (e.g., a pair) or moreuniversal sequences that may be present in different members of acollection of nucleic acid molecules can allow the replication oramplification of multiple different sequences using at least one, two(e.g., a pair) or more single universal primers that are complementaryto the universal sequences. Thus, a universal primer includes a sequencethat can hybridize specifically to such a universal sequence. The targetnucleic acid sequence-bearing molecules may be modified to attachuniversal adapters (e.g., non-target nucleic acid sequences) to one orboth ends of the different target sequences, the adapters providingsites for hybridization of universal primers. This approach has theadvantage that it is not necessary to design a specific pair of primersfor each template to be generated, amplified, sequenced, and/orotherwise analyzed; a single pair of primers can be used foramplification of different templates provided that each template ismodified by addition of the same universal primer-binding sequences toits 5′ and 3′ ends.

Solid Support

A solid support for use in a number of embodiments that are describedherein refers to any solid surface or collection of solid surfaces towhich nucleic acids can be covalently attached, for example, latexbeads, dextran beads, polystyrene, polypropylene, polyacrylamide gel,gold surfaces, glass surfaces, optical fibers, and silicon wafers, orother materials that are typically solids at ambient room temperaturesand that are amenable to chemical modification to afford covalentlinkage to a nucleic acid. In one embodiment, the preferred solidsupport is a glass surface.

The solid support may comprise a surface that may be contained in a flowchamber such as a flow cell, allowing convenient movement of liquidsacross the surface to enable the transfer of reagents. Exemplary flowcells that can be used in this manner are described in WO 2007/123744,which is incorporated herein by reference in its entirety.

Nucleic Acid Immobilization on Solid Supports

According to certain embodiments contemplated herein, there are providednucleic acids that are immobilized to a solid support. For example,colony primers, template nucleic acids, and/or nucleic acid complements(e.g., a complement of a nucleic acid template) may be attached andthereby immobilized to a solid support as described herein. Any of awide variety of chemical or non-chemical attachment methods may beemployed, including, for example, labels that are capable of binding toa ligand or receptor, or chemically-modifiable functional groups thatcan mediate covalent linkage of a nucleic acid to the solid support,such as the chemically-modifiable functional groups described herein.Immobilization refers to the attachment of nucleic acid to a solidsupport by one or more of a covalent attachment, irreversible passiveadsorption, and specific high-affinity binding interactions betweenmolecules (for example, immobilization of biotinylated molecules on anavidin-coated surface). The attachment is typically of sufficientstrength that it cannot be removed by washing with water or aqueousbuffer under DNA-denaturing conditions. Specific binding interactionstypically refers to conditions where two binding partners remain inphysical association with one another as a consequence of one or moremolecular properties selected from molecular shape complementarity,electrostatic or charge attractions, hydrophobic attractions, stericinteractions, hydrophilic interactions, van der Waals forces, hydrogenbonding, and the like, while structurally unrelated molecules that fail,qualitatively or quantitatively, to share such properties are unable toremain in association with either binding partner.

Nucleic acids such as oligonucleotide primers may be immobilized to asolid support by single point covalent attachment to the surface of thesolid support. The point of attachment can, for example, be situated ator near the 5′ end of the nucleic acid. Any suitable covalent attachmentmeans known in the art may be used for this purpose. The chosenattachment chemistry will depend on the nature of the solid support, andon any derivatization or functionalization applied to it. Anoligonucleotide primer to be immobilized may itself include a chemicalmoiety, which may be a non-nucleotide chemical modification, tofacilitate attachment. For example, the oligonucleotide primer mayinclude a sulphur-containing nucleophile, such as phosphorothioate orthiophosphate, located at the 5′ end. In the case of solid-supportedpolyacrylamide hydrogels, this nucleophile will bind to a bromoacetamidegroup present in the hydrogel. A more particular means of attachingprimers and templates to a solid support is via 5′ phosphorothioateattachment to a hydrogel comprised of polymerized acrylamide andN-(5-bromoacetamidylpentyl) acrylamide (BRAPA), as described in U.S.patent application Ser. No. 10/585,373 and WO 05/065814, each of whichis herein incorporated by reference in its entirety. Other means forattaching a nucleic acid such as an oligonucleotide primer or a templateto a solid support include chemically-modifiable functional groups thatcan mediate covalent linkage of a nucleic acid to the solid support,such as the chemically-modifiable functional groups described herein.Attachment can also occur via ligand-receptor interactions such as thosedescribed elsewhere herein.

Chemically-Modifiable Functional Group

Chemically modifiable functional groups to be added at the 5′ end of thenucleic acids to be immobilized on solid supports may be thiol,hydroxyl, dimethoxyltrityl (DMT), amino, or phosphate groups, as well ascarboxylic or aldehyde moieties. Examples of crosslinking agents usefulto derivatize a solid support are1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),succinic anhydride, phenyldiisothiocyanate or maleic anhydride, or ahetero-bifunctional crosslinker such as, for example,m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl[4-iodoacethyl]aminobenzoate (SIAB), Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC),N-y-maleimidobutyryloxy-succinimide ester (GMBS),Succinimidyl-4-[p-malei-midophenyl]butyrate (SMPB) and the sulfo(water-soluble) corresponding compounds. In a preferred embodiment, thenucleic acid templates and primers are modified with thiol, phosphate oramino group at the 5′ end modification and immobilized using animmobilization solution containing1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) ascrosslinking agent.

In other embodiments, the chemically-modifiable functional group asdescribed herein may refer to a chemical group such as, for example, aphosphate group, a carboxylic or aldehyde moiety, a thiol, or an aminogroup.

Preparation of Nucleic Acid Clusters

Methods of generating nucleic acid clusters for use in high-throughputnucleic acid technologies have been described, as noted above; see, forexample, U.S. Pat. No. 7,115,400, U.S. Patent Application PublicationNos. 2005/0100900 and 2005/0059048, and PCT Publication Nos. WO98/44151, WO 00/18957, WO 02/46456, WO 06/064199, and WO 07/010,251,each of which is incorporated by reference herein in its entirety.

As also noted above, however, currently available nucleic acid clustertechnologies typically involve the generation of randomly andirregularly spaced nucleic acid clusters on a solid support. The failureof the current technologies to resolve overlapping signals from two ormore nucleic acid clusters has led to less dense and lower throughputassays, in order to avoid the difficulties associated with interpretingand resolving the overlapping signals created by abutting, tightlyproximate and/or extremely densely arrayed clusters. In contrast, thecompositions and methods of the presently described inventionembodiments provide, in pertinent part, nucleic acid clusters in whichthe center or origin of the nucleic acid cluster can be distinguishedfrom the surrounding nucleic acids in the cluster, thus affordingcertain related advantages as described herein.

Without wishing to be bound by any particular theory, it is presentlycontemplated that by detectably labeling, with a detectable center labelas described herein, the first strand or stands (i.e., the originaltemplate nucleic acid and/or the complement thereof) of a nucleic acidcluster that is amplified outwardly and radially from a fixed positionon the solid support that is occupied by the original template and/orits complement, the origin or center of the nucleic acid cluster can beidentified. As the center (origin) of the nucleic acid cluster can beidentified by virtue of the signal emanating from the detectable label,the positions of two or more overlapping clusters (e.g., abutting,tightly proximate and/or extremely densely arrayed clusters thatgenerate overlapping signals, such as the signals from fluorescentsequencing labels that otherwise could not be readily assigned to asingle source cluster) can then be resolved. The signal from the labeledorigin (e.g., center, or the substantially central location) of eachcluster will appear as a discrete, diffraction limited spot, and hencewill be focused and geometrically much smaller than a signal generatedfrom a nucleic acid cluster that has been labeled across the entirecluster, which can't reach the diffraction limit. Unlike previouslydescribed nucleic acid clusters, in which incorporation of detectablelabels at locations other than just the centers (origins) can result inambiguity as to the source of a given signal when two or more clustersoverlap on the solid support (e.g., abutting, tightly proximate and/orextremely densely arrayed clusters that generate overlapping signalsthat cannot be assigned to a single discrete source cluster), in thepresent invention embodiments, the detectable center label signals fromthe identifiable centers of the overlapping clusters will not overlap.Such non-overlapping detectable center label signals, even fromotherwise overlapping clusters, may therefore be assigned to a singlediscrete source cluster with high precision.

Accordingly, in particular embodiments the present inventioncontemplates methods to identify and resolve overlapping signalsgenerated from a plurality of nucleic acid clusters on a solid support,such as abutting, tightly proximate and/or extremely densely arrayedclusters. In related embodiments, detectable signals that permitunambiguous identification of the substantially central location in oneor more nucleic acid clusters can be used in concert with, e.g.,sequencing methods that might otherwise result in ambiguity as to thesource of a given sequencing label signal (i.e., a signal that isdifferentiable from the center/origin signal that permits identificationof the identifiable center of each cluster as provided herein) whensignals from two or more clusters overlap on the solid support, or asanother example, when the signals from two or more clusters overlap inan image of the solid support that has been generated using signals fromsequencing labels. These and similar embodiments may be amenable to anumber of high-throughput assay formats, e.g., nucleic acid sequencing,gene expression analysis, epigenetic analysis, genotyping, and others asdescribed herein.

Signals produced by the detectable label at the center (origin) of anucleic acid cluster, and detectable sequencing label signals (i.e., asignal that is differentiable from the center/origin signal that permitsidentification of the identifiable center of each cluster as providedherein, for example and without limitation, as a function of intensity,conductivity, wavelength or other spectral or physicochemicalproperties) may be detected using any suitable apparatus that includesan appropriate detection device, which will be apparent to the skilledartisan as a function of the assay configuration, includinginstrumentation, and of the particular detectable center label that isemployed, and of the particular sequencing label signal (i.e., a signalthat is differentiable from the center/origin signal that permitsidentification of the identifiable center of each cluster), such as alabeled sequencing nucleotide. For instance, a preferred detectionsystem for fluorescent labels is a charge-coupled device (CCD) camera,which can optionally be coupled to a magnifying device, for example amicroscope. Using such technology it is possible to simultaneouslymonitor many colonies in parallel. A confocal scanner (e.g.,laser-scanning confocal microsopy) can also be used to detect signals. Alabel that is used to identify a central location of a cluster may beretained throughout a sequencing process, or may be removed beforesequencing commences. In cases where the label is removed prior tosequencing, it can be advantageous to use a label that is the same as orsimilar to one or more of the labels used in the sequencing steps.

Immobilized nucleic acid clusters may be designated as indistinct oroverlapping when the amplification and/or sequencing and/or othernon-center label signals detected from each cluster (i.e., a signal thatis differentiable from the center/origin signal that permitsidentification of the identifiable center of each cluster as disclosedherein) share about 0.5%, about 1%, about 2.5%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 75%, about 90%, about 95%, or about 99% of thesame area, for example on a solid support. Another measure is thepercentage of the perimeter of a first cluster that is in apparentcontact with an abutting cluster, and which can be, for example, atleast about 0.5%, about 1%, about 2.5%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 75%, about 90%, about 95%, or about 99% of the perimeter ofthe first cluster. In another embodiment, the positions of labeledsequencing nucleotides that comprise non-center labels which areincorporated into a nucleic acid molecule as provided herein during asequencing reaction overlap if the signals detected from each labeledsequencing nucleotide (e.g., a sequencing label signal such as a signalthat is differentiable from the center/origin signal that permitsidentification of the identifiable center of each cluster, such as alabeled sequencing nucleotide) share about 0.5%, about 1%, about 2.5%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 75%, about 90%, about 95%,or about 99% of the same area.

It will be appreciated that while the shared area can be the actualphysical area on a solid support, shared area may additionally oralternatively comprise the area shared in a graphical representation ofthe signal detected from each of the detectably labeled clusters orsequencing nucleotides, such as a representation produced by anycommonly used image analysis software. Thus, the apparent overlap can bea function of the resolution of the detection system that is used toimage adjacent, abutting or overlapping clusters.

Embodiments disclosed herein provide methods for producing at least onenucleic acid cluster having an identifiable center. In particularembodiments, at least 0.5, at least 1, at least 2, at least 5, at least10, at least 25, at least 50, at least 100, at least 500, at least 1000,at least 10,000 at least 100,000, at least 1,000,000, or at least10,000,000 or more nucleic acid clusters per square millimeter aregenerated on a solid support. In other various embodiments, compositionsare provided that comprise one or more nucleic acid clusters each havingan identifiable center.

Nucleic acid clusters can be generated from a single nucleic acidtemplate as provided herein. In one embodiment, a single nucleic acidtemplate may be immobilized at it 5′ end to a solid support. In anotherembodiment, a single nucleic acid template may be immobilized throughbase-pairing or hybridization to an oligonucleotide primer that isimmobilized, for example, at its 5′ end to a solid support. The presentmethods contemplate the simultaneous production of one or a plurality ofnucleic acid clusters, each of which may contain different nucleic acidtemplates. As used herein, the term “plurality” means more than one. “Aplurality of copies” of nucleic acids, or “a plurality of nucleic acidclusters”, refers to at least or more than 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 100, 500, 1000, 10,000, 100,000, 1,000,000,or 10,000,000 copies or nucleic acid clusters. The skilled artisan willappreciate that any intervening number of copies or clusters is alsocontemplated.

According to certain embodiments, a plurality of nucleic acid templatesmay comprise the nucleic acids to be amplified (e.g., target nucleicacids), wherein the nucleic acids contain at their 5′ ends anoligonucleotide sequence Y and at the 3′ end an oligonucleotide sequenceZ. In addition, the nucleic acid(s) carry at the 5′ end a functionalgroup, as described herein and as known in the art, which allows thenucleic acid(s) to be immobilized on a solid support. In certainembodiments, a plurality of colony primers X, which can hybridize to theoligonucleotide sequence Z, carries at the primer 5′ ends a functionalgroup that allows the primers X to be immobilized to a solid support. Ina preferred embodiment, there is provided a mixture comprising aplurality of nucleic acid templates and colony primers that arecovalently bound to a solid support at their 5′ ends. In a distinct butrelated embodiment, a mixture is provided comprising a plurality ofnucleic acid templates that are immobilized by hybridization toimmobilized colony primers, which are covalently bound to a solidsupport at their 5′ ends. In another embodiment, pluralities of twodifferent colony primers X may be mixed with a plurality of nucleic acidtemplates.

Preferably, the sequences of the colony primers X are such that theoligonucleotide sequence Z can hybridize to one of the colony primers Xand the oligonucleotide sequence Y is the same as another one of thecolony primers. In another embodiment, the oligonucleotide sequence Zmay be complementary to oligonucleotide sequence Y, (Y′), and all of theprimers X have the same sequence as oligonucleotide sequence Y. Inanother embodiment, the plurality of colony primers X may comprise adegenerate primer sequence and the plurality of nucleic acid templatescomprise the nucleic acids to be amplified and do not containoligonucleotide sequences Y or Z at the 5′ and 3′ ends respectively.

In certain embodiments, an oligonucleotide sequence Z is complementaryto an oligonucleotide sequence Y and an immobilized oligonucleotideprimer X comprises a sequence that is substantially identical tooligonucleotide sequence Y. In certain other embodiments, the solidsupport comprises a first and a second immobilized oligonucleotideprimer X which are different from each other, wherein theoligonucleotide sequence Z can hybridize to the first immobilizedoligonucleotide primer X and the second immobilized oligonucleotideprimer X has a sequence that is substantially identical tooligonucleotide sequence Y. One of skill in the art will appreciate thatthe nucleic acid sequences of oligonucleotide sequences Y and Z, and ofone or more colony primers X, are designed such that they hybridizespecifically to their complementary sequences and display negligiblebinding to non-specific sequences.

In view of the present disclosure the skilled person will appreciatethat in related embodiments there may be employed any number ofdifferent combinations of universal, specific, and/or degenerateoligonucleotide primers, to achieve a particular desired configurationfor producing one or a plurality of nucleic clusters, each having anidentifiable center, as provided herein.

In a preferred embodiment, the 5′ ends of colony primers X of theinvention are modified to carry a functional group to covalently attachthe primers to the solid support. In addition, in certain embodiments,the colony primers can be designed to include additional desiredsequences such as, for example, restriction endonuclease sites or othertypes of cleavage sites each as ribozyme cleavage sites or chemicalcleavage sites. Such sites are advantageous for rendering clusterssingle-stranded and then allowing subsequent regeneration of the clusterfor analysis of the complementary strand. Other desirable sequencesinclude, but are not limited to, fold-back DNA sequences (which formhairpin loops or other secondary structures when renderedsingle-stranded), “control” DNA sequences which direct a protein/DNAinteraction, such as for example a promoter DNA sequence which isrecognized by a nucleic acid polymerase or an operator DNA sequencewhich is recognized by a DNA-binding protein.

Immobilization of a colony primer X to a support by the 5′ end leavesits 3′ end free from the support such that the colony primer isavailable for chain extension by a polymerase once hybridized to acomplementary oligonucleotide sequence contained at the 3′ end of thenucleic acid template.

Nucleic acid templates and colony primers that each have an immobilizingfunctional group at their 5′ end may be mixed together in appropriateproportions so that when they are immobilized to the solid support anappropriate density of immobilized nucleic acid templates and colonyprimers is obtained. The proportion of colony primers in the mixture maybe higher than the proportion of nucleic acid templates, or the ratio ofcolony primers to nucleic acid templates may be such that when thecolony primers and nucleic acid templates are immobilized to the solidsupport, a “lawn” of colony primers is formed comprising a plurality ofcolony primers being located at an approximately uniform density overthe whole or a defined area of the solid support, with one or aplurality of nucleic acid templates being immobilized individually atirregular intervals within the lawn of colony primers.

Nucleic acid templates may be provided in single stranded form or indouble stranded form with one or both 5′ ends modified so as to allowdirect immobilization to the support. After completion of the attachmentprocess, the strands may desirably be separated, for example, by heatingto 94° C. or by using a suitable chemical denaturant (e.g., about 0.1 toabout 0.5N NaOH), before washing the released strands away. It will beappreciated that in the case where both strands of the double strandednucleic acid molecules have reacted with the surface and are bothattached at their 5′ ends, the result will be the same as in the casewhen only one strand is attached and one amplification or extension stephas been performed. In other words, in the case where both strands of adouble stranded template nucleic acid have been immobilized to a solidsupport, both strands are necessarily immobilized close to each otherand are indistinguishable from the result of immobilizing only onestrand and performing one amplification or extension step. Thus, singlestranded and double stranded template nucleic acids might be used forproviding template nucleic acids attached to the surface and aresuitable for nucleic acid cluster generation.

In certain other embodiments, nucleic acid templates may be provided insingle stranded form or in double stranded form with no modified 5′end(s); in which case, the nucleic acid templates can be indirectlyimmobilized to a solid support by hybridization to an immobilized colonyprimer. The single stranded and/or double stranded template nucleicacids may, according to various other contemplated embodiments, compriseone or more detectable labels, as described elsewhere herein.Immobilization of a detectably labeled nucleic acid template provides anidentifiable center for any cluster amplified or formed therefrom. Adouble stranded nucleic acid template having both strands that areimmobilized to a solid support may comprise one or more detectablelabels, or alternatively, either one of the strands of the doublestranded nucleic acid template that is immobilized to a solid supportmay comprise one or more detectable labels. Detectable labels can beadded to template nucleic acids by any of a variety of methods set forthherein including, but not limited to, amplification using labeledprimers and/or nucleic acids. The amplification can be carried out insolution or on solid phase prior to immobilizing the amplificationproducts as templates on a solid phase surface where clusters will beformed.

The distance between the individual colony primers and/or the individualnucleic acid templates (and hence the density of the colony primersand/or nucleic acid templates) can be controlled by altering theconcentration of colony primers and nucleic acid templates that aredirectly immobilized to the support. A preferred density of colonyprimers is at least 1 fmol/mm², preferably at least 10 fmol/mm², andmore preferably between 30 to 60 fmol/mm². The density of nucleic acidtemplates for use in the method of the invention is typically 10,000/mm²to 100,000/mm². Higher densities are also achievable, for example,100,000/mm² to 1,000,000/mm² and 1,000,000/mm² to 10,000,000/mm².

Controlling the density of nucleic acid templates and colony primersdirectly immobilized to the solid support allows the final density ofnucleic acid clusters on the surface of the support to be controlled.This is due to the fact that according to the methods described herein,one nucleic acid cluster can result from the attachment of one nucleicacid template. The density of nucleic acid molecules within a singlenucleic acid cluster can also be controlled by controlling the densityof colony primers that are attached to the solid support. In embodimentswherein the nucleic acid templates are indirectly immobilized to thesolid support, e.g., by hybridization to an immobilized colony primer,the concentration and duration of hybridization, as well as the numberof hybridizations performed may be varied to control the density of thenucleic acid clusters generated on the solid support. Advantageously,the compositions and methods of the present embodiments provide higherdensities of immobilized nucleic acids and higher-throughput analysesthan could previously be practiced conveniently, by virtue of producingclusters having an identifiable center.

Thus, in a preferred embodiment, the present invention contemplates acomposition comprising a solid support comprising one or moreimmobilized primers; and at least one template nucleic acid that iseither directly immobilized at its 5′ end to the solid support orindirectly immobilized to the solid support by hybridization to one ofthe one or more immobilized primers, wherein either or both of theprimer and the template comprise a detectable label that may be used asa detectable center label as provided herein. In particular embodiments,the primer or the template that comprise a detectable label can be partof a cluster in which other nucleic acids of the cluster have the samesequence but do not have the detectable label.

Preparation of Nucleic Acid Templates

A library of nucleic acid templates may be prepared using techniqueswhich are standard or conventional in the art. Generally these will bebased on general molecular biology techniques. The nucleic acids to beused as templates can be obtained using methods well known anddocumented in the art. For example, by obtaining a nucleic acid from abiological sample as provided herein, such as total DNA, genomic DNA,cDNA, total RNA, mRNA, or cRNA, a library of nucleic acid templates canbe prepared, for instance, by generating fragments therefrom by limitedrestriction enzyme digestion and/or by mechanical means (e.g.,subjecting to shearing force).

Typically, the nucleic acid to be used as a template is first obtainedin double stranded form. When the nucleic acid is provided in singlestranded form, as mRNA for example, it may be first made into a doublestranded form by means well known and documented in the art, forexample, by synthesizing a cDNA using oligo-dT primers, reversetranscriptase, and DNA polymerase. The nucleic acid to be used as atemplate may obtained in double stranded form of appropriate length andused without further modifications as a nucleic acid template.

The nucleic acid to be used as a template may, for example, be obtainedin double stranded form of appropriate length, and oligonucleotidesequences corresponding to specified oligonucleotide sequences Y and Zmay be joined to each end, i.e., to both the 5′ and 3′ ends of thenucleic acid sequence, to form a nucleic acid template. This can be doneusing methods which are well known and documented in the art, forexample by ligation, or by inserting the nucleic acid to be amplifiedinto a biological vector at a site which is flanked by the appropriateoligonucleotide sequences. Alternatively, if at least part of thesequence of the nucleic acid to be amplified is known, the nucleic acidtemplate containing oligonucleotide sequences Y and Z at the 5′ and 3′ends respectively, may be generated by polymerase chain reaction (PCR)using appropriate PCR primers which include sequences specific to thenucleic acid to be amplified and the primer sequences, Y and Z. Beforeattaching the nucleic acid template to the solid support, it can be madeinto a single stranded form using methods which are well known anddocumented in the art, for example by heating to approximately 94° C.and quickly cooling to 0° C. on ice.

The oligonucleotide sequence contained at the 5′ end of the nucleic acidmay be of any sequence and any length and is denoted herein as sequenceY. Suitable lengths and sequences of oligonucleotide may be selectedusing methods well known and documented in the art, and as describedelsewhere herein. For example the oligonucleotide sequences attached toeach end of the nucleic acid to be amplified are normally relativelyshort nucleotide sequences of about 5, about 10, about 15, about 20,about 25, about 50, about 75, or about 100 nucleotides in length. Theoligonucleotide sequence contained at the 3′ end of the nucleic acid canbe of any sequence and any length and for use in certain embodiments isdenoted herein as sequence Z. Suitable lengths and sequences ofoligonucleotide can be selected using methods well known and documentedin the art, and as described elsewhere herein. For example theoligonucleotide sequences contained at each end of the nucleic acid tobe amplified are normally relatively short nucleotide sequences of about5, about 10, about 15, about 20, about 25, about 50, about 75, or about100 nucleotides in length.

The sequence of the oligonucleotide sequence Z may be such that it canhybridize to one of the colony primers X. In a preferred embodiment, thesequence of the oligonucleotide sequence Y is such that it issubstantially identical to another of the colony primers. In anotherpreferred embodiment, the oligonucleotide sequence Z is complementary tooligonucleotide sequence Y and all of the colony primers X have asequence substantially identical to oligonucleotide sequence Y.

When producing nucleic acid templates according to certain hereindisclosed embodiments, additional desirable sequences may optionally beintroduced by methods well known and documented in the art. Suchadditional sequences may include, for example, restriction enzyme sites,or certain nucleic acid tags in order to permit identification ofamplification products of a given nucleic acid template sequence. Otherexamples of desirable sequences may include fold-back DNA sequences(which form hairpin loops or other secondary structures when renderedsingle-stranded), “control” DNA sequences which direct protein/DNAinteractions, such as for example a promoter DNA sequence which isrecognized by a nucleic acid polymerase, or an operator DNA sequencewhich is recognized by a DNA-binding protein.

If there are a plurality of nucleic acid sequences to be amplified thenthe attachment of oligonucleotides Y and Z can be carried out in thesame or different reaction. Furthermore, persons familiar with the artwill appreciate that template nucleic acids may be prepared frommultiple sources and analyzed simultaneously. Each sample of templatenucleic acids could be prepared using different adapters oroligonucleotide sequences at the 5′ and 3′ ends. For example, sample 1comprises adapters with the oligonucleotide sequences X and Y, sample 2comprises adapters with the oligonucleotide sequences A and B, sample 3comprises adapters with the oligonucleotide sequences D and E, and soforth. Once template nucleic acids have been prepared from samples 1-3,they can be mixed and combined with one or two colony primers X(specific to sample 1), one or two colony primers C (specific to sample2), and one or two colony primers F (specific to sample 3).

In yet another embodiment, more than two, for example, three, four, ormore than four different colony primers may be immobilized to the solidsupport, for instance, using any of the nucleic acid immobilizationstrategies described herein or known to the art. In this manner morethan one library of nucleic acid templates, prepared from differentpatients, for example, and with common sequences (e.g., universal primerbinding sites) that differ between the libraries (wherein commonsequences attached thereto are specific for each library), can beanalyzed using the methods of the present invention.

Particularly useful methods for preparing nucleic acid templates aredescribed, for example, in Bentley et al., Nature 456:49-51 (2008) andUS 2007/0128624, each of which is incorporated herein by reference. Inparticular embodiments, methods of preparing nucleic acid templates,such as those set forth above or elsewhere herein, can be carried outusing labeled primers and/or labeled nucleotides in order to producetemplates having labels useful for producing an identifiable center fora cluster.

In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples oftemplate nucleic acids from the same, similar, or different sources maybe analyzed simultaneously using the compositions and methods of thepresent invention.

In particular embodiments, a nucleic acid template that has beenprepared can be amplified before further use in certain presentlydisclosed methods. Such amplification may be carried out using methodswell known and documented in the art, for example by inserting thetemplate nucleic acid into an expression vector and amplifying it in asuitable biological host, or amplifying it by PCR. This a prioriamplification step is not, however essential, as certain of the methodsdescribed herein, e.g., for producing or annotating nucleic acidclusters having identifiable centers, comprise amplification steps bywhich multiple copies of the nucleic acid template may be produced in anucleic acid cluster generated from a single copy of the nucleic acidtemplate.

It will be appreciated that template nucleic acids as described hereinmay, in particular embodiments, comprise one or more detectable labelsas described elsewhere herein. The detectable label(s) may be at the 5′end, 3′ end, and/or at any position along the length of the templatenucleic acid.

Cluster Formation: Primer Extension and Nucleic Acid Amplification

As used herein, the term “solid-phase amplification” refers to a nucleicacid amplification conducted on a solid support. In particularembodiments, amplification is geometric. Solid-phase amplification maycomprise a nucleic acid amplification reaction comprising only onespecies of oligonucleotide primer immobilized to a solid support.Alternatively, the solid support may comprise a plurality of first andsecond different immobilized oligonucleotide primer species. Solid-phaseamplification refers to any nucleic acid amplification reaction carriedout on or in association with a solid support such that all or a portionof the amplified nucleic acids remain immobilized on the solid supportas they are formed. In particular, the term encompasses solid phaseamplification reactions analogous to standard solution phase primerextension or PCR except that one or both of the forward and reverseamplification primers is/are immobilized on the solid support.

Solid phase nucleic acid amplification reactions may generally compriseat least one of two different types of nucleic acid amplification,interfacial and surface (or bridge) amplification. For instance, ininterfacial amplification the solid support comprises a nucleic acidtemplate that is indirectly immobilized to the solid support byhybridization to an immobilized colony primer X as described herein, theimmobilized colony primer may be extended in the course of apolymerase-catalyzed, template-directed elongation reaction (e.g.,primer extension) to generate an immobilized complementary nucleic acidthat remains attached to the solid support. After the extension phase,the nucleic acids (e.g., template and its complementary product) may bedenatured such that the template nucleic acid is released into solutionand made available for hybridization to another colony primer X. Theindirectly immobilized nucleic acid may be made available in 1, 2, 3, 4,5 or more rounds of primer extension or may be washed out of thereaction after 1, 2, 3, 4, 5 or more rounds of primer extension.

The second type of amplification, surface (or bridge) amplification,occurs when, on a solid support, an immobilized nucleic acid template ornucleic acid complement hybridizes to an immobilized colony primer X.The 3′ end of the immobilized nucleic acid template or immobilizednucleic acid complement provides the template for apolymerase-catalyzed, template-directed elongation reaction (e.g.,primer extension) extending from the complementary and immobilizedcolony primer X. The resulting double-stranded product “bridges” the twocolony primers, and both strands are covalently attached to the support.In the next cycle, following denaturation that yields a pair of singlestrands (the immobilized template and the extended-primer product)immobilized to the solid support, both immobilized strands can serve astemplates for new primer extension, thereby providing a mechanism fornucleic acid cluster formation.

“Nucleic acid amplification” includes the process of amplifying orincreasing the numbers of a nucleic acid template and/or of a complementthereof that are present, by producing copies of the template and/or orits complement. The amplification process may, for example, be anexponential, geometric or linear solid phase amplification. Inexponential amplification, the number of nucleic acid copies increasesat an exponential rate. For example, in an ideal PCR reaction with 30cycles, 2 copies of template DNA will yield 2³° or 1,073,741,824 copies.In linear amplification the number of nucleic acid copies increases at alinear rate. For example, in an ideal 4-hour linear amplificationreaction whose copying rate is 2000 copies per minute, one molecule oftemplate DNA will yield 480,000 copies. Geometric amplification, asanother example, is spatially determined and the rate of amplificationis between that of exponential and linear (Mercier and Slater.Biophysical Journal. Volume 89; July 2005; pp. 32-420).

Nucleic acid amplification may involve several components: a targetnucleic acid molecule (e.g., the nucleic molecule to be amplified suchthat more copies of it are obtained), a molar excess of one or moreamplification oligonucleotide primers which bind to the target nucleicacid molecule by specific hybridization, deoxyribonucleosidetriphosphates (dATP, dTTP, dCTP and dGTP) and a polymerase enzyme.Various types of nucleic acid amplification contemplated by the presentmethods include primer extension, thermal polymerase chain reaction, andisothermal polymerase chain reaction, but the embodiments contemplatedherein are not intended to be so limited and may also include othernucleic acid amplification strategies and methodologies as are known inthe art to be compatible with the compositions and methods of thepresent disclosure.

Primer Extension

“Primer extension” includes the extension of one or more immobilizedcolony oligonucleotide primers X by polymerase-driven serial addition tothe primer of one or more nucleotides as directed by a nucleic acidtemplate according to nucleotide base-complementarity rules, when theprimer(s) is hybridized to the nucleic acid template. In certainpreferred embodiments described herein, the first nucleic acidcomplement of a template in a nucleic acid cluster may be generated by aprimer extension reaction. Primer extension thus comprises the 5′ to 3′extension of an oligonucleotide primer hybridized to a target nucleicacid. Primer extension may be performed using, but does not require,thermocycling (e.g., temperature fluctuations, usually controlled, thatpermit alternating periods of temperatures conducive to nucleic acidhybridization and temperatures conducive to denaturation of nucleic acidduplexes, typically in the presence of a thermostable polynucleotidepolymerase) and may also be conducted at static (e.g., substantiallyinvariant or isothermal) temperature. For example, when a DNA dependentpolymerase such as Taq DNA polymerase is used for primer extension, theprimer extension may be conducted at a temperature of 72° C. In anothernon-limiting example, primer extension may be carried out using areverse transcriptase at a constant temperature of 42-50° C.

Thermal Polymerase Chain Reaction

A thermal PCR reaction includes one or a plurality of repeatedtemperature cycling steps e.g., amplification cycles characterized byalternating periods of temperatures conducive to nucleic acidhybridization and temperatures conducive to denaturation of nucleic acidduplexes, typically in the presence of a thermostable polynucleotidepolymerase) that may be performed to accomplish the linear orexponential amplification of a nucleic acid. Generally thermal PCR isconducted using a plurality of nucleic acid duplexes that each compriseone or more primers annealed to complementary strands of a dsDNAtemplate that has been denatured (melted apart to yield single strandnucleic acid, e.g., ssDNA) at high temperature (90° C. to 100° C.).Nucleic acid amplification of one or a plurality of copies of animmobilized nucleic acid template and/or of an immobilized nucleic acidcomplement of a template, such as at a substantially central locationwithin a nucleic acid cluster as provided herein, may be performed usingone or more oligonucleotide primers having their 5′ ends immobilized toa solid support. Each amplification cycle comprises a melting,annealing, and polymerase-driven, template-directed (orcomplement-directed) extension step usually carried out at differingtemperatures in the presence of a thermostable polynucleotidepolymerase, nucleotide precursors to be incorporated by extension, andco-factors and ancillary reagents as will be familiar to those skilledin the art.

Isothermal Polymerase Chain Reaction

Isothermal polymerase chain reaction refers to PCR in which thetemperature of a system remains constant: e.g., ΔT=0. This typicallyoccurs when a reaction system is present in a reaction vessel that is incontact with an outside thermal reservoir (for example, heat baths andthe like), and the processes occur slowly enough to allow the system tocontinually adjust to the temperature of the reservoir through heatexchange.

The term “substantially isothermal” as used herein is therefore intendedto mean that the system is maintained at essentially the sametemperature during the reaction. The term is also intended to captureminor deviations in temperature which might occur as the systemequilibrates, for example when components which are of lower or highertemperature are added to the system. Thus, it is intended that the termincludes minor deviations from the temperature initially chosen toperform the method and those in the range of deviation of commercialthermostats. More particularly, the temperature deviation will be nomore than about +/−2° C., more particularly no more than about +/−1° C.,yet more particularly no more than about +/−0.5° C., no more than about+/−0.25° C., no more than about +/−0.1° C. or no more than about+/−0.01° C. Isothermal amplification methods are well known in the artof solid phase nucleic acid cluster formation, for example, as describedin U.S. patent application Ser. No. 11/725,597, which is hereinincorporated by reference in its entirety.

The skilled artisan will appreciate that the initial primer extensionproduct may comprise one or more detectable labels. In addition, one ormore rounds of primer extension may be conducted under conditionswherein in each round one or a plurality of detectably labelednucleotides may be incorporated into the primer extension products. Anextension and/or amplification reaction may comprise subjecting thesolid support having one or more attached nucleic acid templates and/orcomplements thereof, and colony primers, to conditions which induceprimer hybridization, for example, by subjecting them to a temperatureof around 65° C. Under these conditions an oligonucleotide sequence Z atthe 3′ end of the nucleic acid template will hybridize to thecomplementary immobilized colony primer X. Then, under conditions and inthe presence of reagents that support primer extension, for example atemperature of around 72° C., the presence of a nucleic acid polymerasesuch as a DNA dependent DNA polymerase or a reverse transcriptasemolecule (e.g., an RNA dependent DNA polymerase), or an RNA polymerase,plus a supply of nucleoside triphosphate molecules or other nucleotideprecursors, for example modified nucleoside triphosphate molecules ornucleoside triphosphates that comprise one or more detectable labels,the colony primer will be extended by the addition of nucleotidescomplementary to the template nucleic acid sequence.

Examples of nucleic acid polymerases which can be used in these andrelated embodiments include DNA polymerase (Klenow fragment, T4 DNApolymerase), heat-stable DNA polymerases from a variety of thermostablebacteria (such as Taq, VENT, Pfu, Tth, Tfl DNA polymerases) as well astheir genetically modified derivatives (TaqGold™, VENTexo, Pfu exo) andother polymerases, such as Bst polymerase. A combination of 1, 2, 3, 4,5, or more polymerase may be used in extension and/or amplificationsteps as described herein to generate a nucleic acid cluster on a solidsupport. A combination of RNA polymerase and reverse transcriptase mayalso be used for amplification of a DNA colony. Preferably the nucleicacid polymerase used for colony primer extension is stable underthermocycling PCR reaction conditions, i.e., repeated cycles of heatingand cooling, and is stable at the denaturation temperature used, usuallyapproximately 94° C. Preferably, in thermocyling nucleic acidamplification reactions the DNA polymerase used is Taq DNA polymerase.In a preferred embodiment, isothermal nucleic acid amplification isused, preferably using Bst or Klenow DNA polymerases

Preferably the nucleoside triphosphate molecules used aredeoxyribonucleotide triphosphates, for example dATP, dTTP, dCTP, dGTP,or are ribonucleoside triphosphates for example dATP, dUTP, dCTP, dGTP.The nucleoside triphosphate molecules may be naturally or non-naturallyoccurring. In a preferred embodiment, the initial primer extensionreaction comprises one or a plurality of detectably labeled nucleosidetriphosphates or deoxyribonucleoside triphosphates.

Accordingly there is provided herein a method for producing at least onenucleic acid cluster having an identifiable center. In relatedembodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 100, 500, 1000, 10,000, 100,000, 1,000,000, or 10,000,000nucleic acid clusters having identifiable centers may be produced. Theforegoing numbers of clusters can be generated per cm² or per mm²depending on the particular analysis, configuration, or other parameterthat may be desired.

Particularly useful methods of isothermal amplification are described,for example, in US 2008/0009420, which is incorporated herein byreference. Useful devices and methods for carrying out isothermalamplification are described, for example, in WO 08/002,502, which isincorporated herein by reference.

As described herein there is thus provided a method for producing atleast one nucleic acid cluster having an identifiable center, whichmethod comprises generating, on a solid support, at least oneimmobilized nucleic acid complement of at least one nucleic acidtemplate, wherein the at least one nucleic acid template and/or the atleast one nucleic acid complement comprises one or more detectablelabels; and amplifying the at least one nucleic acid template and/or theat least one nucleic acid complement to obtain on the solid support atleast one nucleic acid cluster, wherein each cluster comprises (a) asubstantially central location comprising the at least one nucleic acidtemplate and/or the at least one nucleic acid complement, and (b) asurrounding region comprising one or more immobilized copies of the atleast one nucleic acid template and/or of the at least one nucleic acidcomplement, and thereby producing the at least one nucleic acid clusterhaving an identifiable center.

Nucleic acid clusters generated as described herein may comprise anidentifiable center comprising at least one detectably labeledimmobilized template nucleic acid and/or at least one detectably labeledimmobilized nucleic acid complement, either or both of which maycomprise a detectable center label as provided herein. The signalgenerated from these detectably labeled nucleic acids indicates asubstantially central location or center of the cluster. The center of acluster may be distinguished from the one or more immobilized copies ofthe at least one nucleic acid template and of the at least one nucleicacid complement that are present in the surrounding region, becausethese surrounding region copies lack the detectable label (e.g., adetectable center label) of the substantially central location of thecluster. In other embodiments, the center of a cluster may bedistinguished from the one or more immobilized copies of the at leastone nucleic acid template and/or the at least one nucleic acidcomplement that are present in the surrounding region, because thesecopies comprise a detectably different detectable label compared to thatwhich is present in the substantially central location of the cluster(e.g., a detectable center label) or due to quenching or masking of anylabel present in the one or more immobilized copies of the at least onenucleic acid template and of the at least one nucleic acid complementthat are present in the surrounding region. In other embodiments, thecenter of a cluster lacks a detectable label and therefore may bedistinguished from the one or more immobilized copies of the at leastone nucleic acid template and of the at least one nucleic acidcomplement in the surrounding region, because these copies comprise adetectable label.

Without wishing to be bound by any particular theory, the detectablelabel as provided herein, which in certain embodiments may be adetectable center label, is stable, such that the label can remaindetectable throughout the process of nucleic acid cluster generation andduring any subsequent analysis. In particular embodiments, antioxidantsare used in the amplification mix to increase the amount of time thatsuch a detectable label remains detectable. These antioxidants may bereplenished throughout the methods of generating nucleic acid clustersdescribed herein and during any subsequent analyses. These and relatedembodiments contemplate, in part, that a detectable label will remainsubstantially detectable for about 1 hour, about 2 hours, about 4 hours,about 6 hours, about 12 hours, about 24 hours, or about 48 or morehours, under the conditions typically employed to practice the presentlydisclosed methods. In addition, it would be understood by those havingskill in the art that stable detectable labels are known (e.g.,radiolabels, fluorescent labels) in the art, for example, as describedin Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor),Plenum Pub Corp, 2nd edition (July 1999) and the Sixth Edition of theMolecular Probes Handbook by Richard P. Haugland; Welch et al. (Chem.Eur. J. 5(3): 951-960, 1999); Zhu et al. (Cytometry 28:206-211, 1997);Prober et al. (Science 238:336-341, 1987); Connell et al. (BioTechniques5(4):342-384, 1987); Ansorge et al. (Nucl. Acids Res. 15(11):4593-4602,1987); and Smith et al. (Nature 321:674, 1986). Thus, in variousembodiments, the extension/amplification steps can be repeated one or aplurality of times under conditions and for a time sufficient for thedetectable label to remain detectable at the substantially centrallocation in the at least one nucleic acid cluster.

Producing Nucleic Acid Clusters having Identifiable Centers

Certain preferred methods for producing one or more nucleic acidclusters having an identifiable center comprise generating at least onedetectably labeled immobilized nucleic acid complement by primerextension as described above, using nucleosides or nucleotide precursorsthat comprise one or more detectable labels, which may be incorporatedinto the extension product. Accordingly, following primer extension, animmobilized nucleic acid complement has been generated, which comprisesat least one detectable label and is complementary to the initialnucleic acid template. Without wishing to be bound by theory, thelocation of this labeled complementary nucleic acid is believed toidentify the origin or center of the cluster as it is the first strandfrom which the rest of the cluster is produced. The second cycle ofextension can also utilize labeled nucleotides, in which case theinitial strands in both ‘orientations’ are labeled. Upon separating theimmobilized complement and the nucleic acid template, for example, byheating or chemical denaturation, one or two immobilized nucleic acidswill be present. For example, if the nucleic acid template was notimmobilized at its 5′ end to the solid support, then only oneimmobilized nucleic acid would be present after the first round ofprimer extension (e.g., the nucleic acid complement of the template,which complement has been generated by primer extension of animmobilized oligonucleotide primer such as a colony primer). In anotherexample, if the nucleic acid template was immobilized at its 5′ end tothe solid support, then both the immobilized nucleic acid template andthe immobilized nucleic acid complement would be present after the firstround of primer extension. The template may include a label that isuseful for identifying a central location or, alternatively, thetemplate can lack such a label.

It will be understood by those skilled in the art that immobilizedcolony primers can be extended one nucleotide at a time during a primerextension reaction by stopping and then re-initiating the primerextension. Nucleotides comprising reversibly blocked 3′ ends, forexample, as described in U.S. Pat. No. 7,427,673, which is hereinincorporated by reference in its entirety, are useful for stopping andre-initiating primer extension reactions. The step-wise incorporation ofa labeled nucleotide into the immobilized primer may be repeated 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more times. As also noted above, one or aplurality of detectably labeled nucleotides may be incorporated into animmobilized colony primer; these may include labeling one, two, three orall four (or more) nucleotides, and the inclusion of two or moredifferent labeling moieties amongst the detectable labels that arepresent during the step of generating (e.g., by primer extension) isalso contemplated. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides each comprising a detectable label may be incorporated intothe immobilized nucleic acid complement. The nucleic acid sequence of animmobilized and detectably labeled nucleic acid complement may compriseabout 1%, about 5%, about 10%, about 25%, about 50%, about 75%, about90% about 95% or about 100% labeled nucleotides.

Following hybridization and extension steps, the duplexed nucleic acidscan be denatured, which results in immobilized single stranded nucleicacids that are then available to initiate further rounds ofamplification through further cycles of hybridization, extension anddenaturation. Such further rounds of amplification will result in anucleic acid cluster comprising a plurality of immobilized copies of thetemplate nucleic acid and its complementary sequence.

In certain embodiments in which the template nucleic acid is immobilizedon the solid support, the template nucleic acid would not be expected tobe able to hybridize to immobilized colony primers that are located at adistance from the template that is beyond the total length of thetemplate nucleic acid. Therefore, the nucleic acid cluster formed fromsuch an immobilized template may be initially restricted to a relativelylocal region of the solid support, i.e., the area in which the initialtemplate nucleic acid was immobilized. Once more copies of the templatemolecule and its complement have been synthesized by carrying outfurther rounds of amplification, i.e., further rounds of hybridization,extension and denaturation, then the boundary of the nucleic acidcluster being generated may be extended further, advancing howeverincrementally from the initial site.

Once nucleic acid clusters having identifiable centers have beengenerated on a solid support as herein described, certain furtherembodiments contemplate an additional step such as, for example,recording the center position in the cluster or annotating the centerposition of the cluster, for advantageous use as described herein. Forexample by way of illustration and not limitation, nucleic acid clusterscomprising an identifiable center permit the interpretation of otherwiseconfounding data from overlapping clusters (e.g., closely spacedadjacent clusters that may be difficult for image analysis algorithms toresolve), as well as resolution of the origin of signals generated fromthe overlapping clusters.

In order to resolve the origin of the nucleic acid cluster, a signalemanating from the detectably labeled immobilized nucleic acid templateand/or the detectably labeled nucleic acid complement of the templatemay be detected by an apparatus that comprises an appropriate detectiondevice, and recorded on a computer memory using image analysis software.A preferred detection system for fluorescent labels is a charge-coupleddevice (CCD) camera, which can optionally be coupled to a magnifyingdevice, for example a microscope. Using such technology it is possibleto simultaneously monitor many clusters in parallel. For example, usinga microscope with a CCD camera and a 10× or 20× objective it is possibleto observe clusters over a surface of between 1 mm² and 4 mm², whichcorresponds to monitoring between 10,000 and 200,000 colonies inparallel. Moreover, this number will increase with improved optics andlarger chips.

An alternative method of monitoring signals emanating from detectablelabels present in the solid phase immobilized nucleic acid clustershaving identifiable centers and produced as described herein is to scanthe surface of the solid support that is substantially covered withclusters. For example, systems may be used in which up to 100,000,000 ormore clusters are arrayed and can be simultaneously monitored by takingpictures with the CCD camera over the whole surface.

Any other devices allowing detection and preferably quantitation of thesignal produced by a detectable label (e.g., a fluorescence signal) thatmay be present on the surface of a solid support may be used to monitorthe nucleic acid clusters having identifiable centers as describedherein. For example, fluorescent imagers or confocal microscopes areknown and may be used for this purpose. If the detectable labels areradioactive, then a detection system capable of monitoring theappropriate radionuclide decay may be selected according to criteriawith which the skilled person will be familiar.

In certain embodiments there is contemplated, in pertinent part, acomposition comprising a solid support and one or more nucleic acidclusters having identifiable centers as described herein, eachcomprising a plurality of nucleic acids having the same sequence andbeing immobilized to the solid support, wherein each nucleic acidcluster comprises an identifiable center comprising a firstsubpopulation of the plurality of nucleic acids that is surrounded by asecond subpopulation of the nucleic acids, wherein each of the nucleicacids in the first subpopulation comprises one or more detectable labelsthat distinguish the nucleic acids in the first subpopulation from thenucleic acids in the second population. These and other nucleic acidclusters comprising an identifiable center will find uses in nucleicacid sequence analysis, gene expression analysis, genotyping of subjectsor biological samples, and the like, as also discussed elsewhere herein.

Annotating the Center of a Nucleic Acid Cluster

As also discussed above, according to certain embodiments the presentinvention provides the ability to resolve the signals generated (e.g.,fluorescent signals) from two or more overlapping nucleic acid clusters.Nucleic acid clusters generated by the methods of the present inventioncomprise an identifiable center which identifies a substantially centerlocation of the cluster. A nucleic acid cluster having an identifiableorigin or center is generated by detectably labeling the initial nucleicacid template and or the initial nucleic acid complement of the nucleicacid cluster such that the detectable label emits a signal fromsubstantially the center of the cluster. The signal generated by theidentifiable center is detected as a small discrete spot and will notoverlap with another identifiable center of a different cluster.

It is possible to resolve optical features separated on a surface by adistance approximately half the wavelength of the light used fordetection. For example, when detecting 500 nm wavelength light, it ispossible to resolve features separated by 250 nm or more. In thisexample, nucleic acid clusters of greater than 250 nm in size shouldthus be resolvable using the two known locations obtained from thecentral location of each cluster, and the sequencing of the wholecluster. It is possible to use different optical systems, for examplehigher magnifications to resolve the central locations of clusters. Thesize of the unlabelled region can be any size without affecting theability to resolve clusters. One benefit of the invention, in particularembodiments, is that clusters can be made larger, which gives rise to ahigher signal intensity during sequencing, enabling longer reads, butthe larger clusters, which may overlap with each other, can still beresolved due to the central marker. Once the identifiable centers fromtwo or more clusters have been identified, their center positions can berecorded or annotated in a computer memory. The positions of the signalsgenerated from the nucleic acid clusters can then be compared to theidentifiable center position. The correlation between one of theoverlapping signals and a particular detectably labeled center wouldresolve the overlap and would result in assigning the data generated toa particular cluster. In particular embodiments, a label can be attachedto nucleic acids in a central location via a cleavable linker. Once acentral location has been detected, the label can be removed by cleavageof the linker. Cleavage can occur before or after a first cycle ofsequencing is performed. In the latter case, the label can be attachedvia a linker that is the same as or similar to the linker that attacheslabels used for sequencing and both types of labels can be removedsimultaneously. If desired, labels may be removed by photobleaching suchthat they are not present during subsequent sequencing cycles.

There is also contemplated herein a method for annotating a centerposition of at least one nucleic acid cluster, comprising generating andamplifying a nucleic acid cluster having an identifiable center asdescribed herein; and recording a center position in the at least onenucleic acid cluster by detecting the detectable center label andtherefrom identifying the position of the substantially central locationin the at least one nucleic acid cluster, and thereby annotating thecenter position of the at least one nucleic acid cluster. The detectablylabeled center of the nucleic acid cluster can be identified by a numberof techniques known in the art and described herein. In variousembodiments, the detection system comprises a detection device to detectthe signal emitted by the detectable label(s), and image analysissoftware that processes the data generated from the detection device andstores the data on a physical computer memory, such as a hard drive orremovable disk drive.

Nucleic Acid Sequencing

Certain methods provided herein comprise determining the nucleotidesequence of one or a plurality of central nucleic acid complementsand/or nucleic acid templates that may be present in nucleic acidclusters having identifiable centers. For example, situations may arisewhere two or more nucleic clusters overlap on a solid support (e.g.,where signals from sequencing labels overlap such that assignment of agiven signal to a single discrete source cluster may be difficult) in amanner that impairs the ability to obtain meaningful nucleic acidsequencing information from either cluster, such that any informationretrieved from the overlapping clusters must be discarded. Accordingly,there are contemplated herein methods for assigning nucleotide sequenceinformation to one of the overlapping clusters, based in pertinent parton the herein afforded ability to identify in each cluster the positionof the center of the cluster, such that nucleotide sequence informationobtained for a cluster can be correlated with its proper source cluster.

Sequencing can be carried out using a sequencing-by-synthesis technique,for example a technique wherein nucleotides are added successively to afree 3′ hydroxyl group, typically provided by annealing of anoligonucleotide primer (e.g., a sequencing primer), resulting insynthesis of a nucleic acid chain in the 5′ to 3′ direction. These andother sequencing reactions may be conducted on the herein describedsolid supports bearing nucleic acid clusters having identifiablecenters. The reactions comprise one or a plurality of sequencing steps,each step comprising determining the nucleotide incorporated into anucleic acid chain and identifying the position of the incorporatednucleotide on the solid support. The nucleotides incorporated into thenucleic acid chain may be described as sequencing nucleotides and maycomprise one or more detectable labels as provided herein, where theskilled person will recognize that selection of the particulardetectable label(s) can be made such that the reporter properties of thesequencing label permits its being distinguished from any detectablecenter label(s) as provided herein that may be present. In relatedembodiments, each sequencing step of a sequencing reaction comprisesdetecting the incorporation and recording the position of a detectablylabeled sequencing nucleotide. Detectably labeled sequencing nucleotidesmay comprise detectable labels, including, but not limited to haptens,radionuclides, enzymes, fluorescent, chemiluminescent, and/orchromogenic agents.

The methods described herein can be used in conjunction with a varietyof sequencing techniques. In some embodiments, the process to determinethe nucleotide sequence of a target nucleic acid can be an automatedprocess.

Some embodiments include sequencing by synthesis (SBS) techniques. SBStechniques generally involve the enzymatic extension of a nascentnucleic acid strand through the iterative addition of nucleotidesagainst a template strand. In traditional methods of SBS, a singlenucleotide monomer may be provided to a target nucleotide in thepresence of a polymerase in each delivery. SBS can utilize nucleotidemonomers that have a terminator moiety or those that lack any terminatormoieties. Methods utilizing nucleotide monomers lacking terminatorsinclude, for example, pyrosequencing and sequencing usingγ-phosphate-labeled nucleotides. In methods using nucleotide monomerslacking terminators, the number of different nucleotides added in eachcycle can be dependent upon the template sequence and the mode ofnucleotide delivery. For SBS techniques that utilize nucleotide monomershaving a terminator moiety, the terminator can be effectivelyirreversible under the sequencing conditions used as is the case fortraditional Sanger sequencing which utilizes dideoxynucleotides, or theterminator can be reversible as is the case for sequencing methodsdeveloped by Solexa, Inc., Hayward, Calif., now Illumina, Inc., SanDiego, Calif.). In preferred methods a terminator moiety can bereversibly terminating.

SBS techniques can utilize nucleotide monomers that have a label moietyor those that lack a label moiety. Accordingly, incorporation events canbe detected based on a characteristic of the label, such as fluorescenceof the label; a characteristic of the nucleotide monomer such asmolecular weight or charge; a by-product of incorporation of thenucleotide, such as release of pyrophosphate; or the like.

Some embodiments include cycle sequencing which is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in U.S. Pat. No. 7,427,673, U.S. Pat. No. 7,414,116, WO04/018497 and U.S. Pat. No. 7,057,026, the disclosures of which areincorporated herein by reference. This approach is being commercializedby Solexa (now Illumina Inc., San Diego, Calif.), and is also describedin WO 91/06678 and WO 07/123,744 (filed in the United States Patent andTrademark Office as U.S. Ser. No. 12/295,337), each of which isincorporated herein by reference in their entireties. The availabilityof fluorescently-labeled terminators in which both the termination canbe reversed and the fluorescent label cleaved facilitates efficientcyclic reversible termination (CRT) sequencing. Polymerases can also beco-engineered to efficiently incorporate and extend from these modifiednucleotides.

In another exemplary type of SBS, pyrosequencing techniques may beemployed. Pyrosequencing detects the release of inorganic pyrophosphate(PPi) as particular nucleotides are incorporated into the nascent strand(Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P.(1996) “Real-time DNA sequencing using detection of pyrophosphaterelease.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001)“Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11;Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method basedon real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568 and U.S. Pat. No. 6,274,320, thedisclosures of which are incorporated herein by reference in theirentireties). In pyrosequencing, released PPi can be detected by beingimmediately converted to adenosine triphosphate (ATP) by ATPsulfurylase, and the level of ATP generated is detected vialuciferase-produced photons.

In accordance with the methods set forth herein, some or all of thenucleotide monomers that are used for sequencing need not have aterminator moiety. Rather, as is the case with pyrosequencing, severalof the nucleotide monomers can be added to a primer in a templatedirected fashion without the need for an intermediate deblocking step.The nucleotide monomers can contain labels for detection, such asfluorescent labels, and can be used in methods and instruments similarto those commercialized by Solexa (now Illumina Inc.). Preferably insuch embodiments, the labels do not substantially inhibit extensionunder SBS reaction conditions. However, the detection labels can beremovable, for example, by cleavage or degradation. Removal of thelabels after they have been detected in a particular cycle and prior toa subsequent cycle can provide the advantage of reducing backgroundsignal and crosstalk between cycles. Examples of useful labels andremoval methods are set forth herein.

In particular embodiments some or all of the nucleotide monomers caninclude reversible terminators. In such embodiments, reversibleterminators/cleavable fluors can include fluor linked to the ribosemoiety via a 3′ ester linkage (Metzker, Genome Res. 15:1767-1776 (2005),which is incorporated herein by reference). Other approaches haveseparated the terminator chemistry from the cleavage of the fluorescencelabel (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), whichis incorporated herein by reference in its entirety). Ruparel et aldescribed the development of reversible terminators that used a small 3′allyl group to block extension, but could easily be deblocked by a shorttreatment with a palladium catalyst. The fluorophore was attached to thebase via a photocleavable linker that could easily be cleaved by a 30second exposure to long wavelength UV light. Thus, either disulfidereduction or photocleavage can be used as a cleavable linker. Anotherapproach to reversible termination is the use of natural terminationthat ensues after placement of a bulky dye on a dNTP. The presence of acharged bulky dye on the dNTP can act as an effective terminator throughsteric and/or electrostatic hindrance. The presence of one incorporationevent prevents further incorporations unless the dye is removed.Cleavage of the dye removes the fluor and effectively reverses thetermination. Examples of modified nucleotides are also described in U.S.Pat. No. 7,427,673, and U.S. Pat. No. 7,057,026, the disclosures ofwhich are incorporated herein by reference in their entireties.

Additional exemplary SBS systems and methods which can be utilized withthe methods and systems described herein are described in U.S. PatentApplication Publication No. 2007/0166705, U.S. Patent ApplicationPublication No. 2006/0188901, U.S. Pat. No. 7,057,026, U.S. PatentApplication Publication No. 2006/0240439, U.S. Patent ApplicationPublication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S.Patent Application Publication No. 2005/0100900, PCT Publication No. WO06/064199 and PCT Publication No. WO 07/010,251, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize sequencing by ligation techniques. Suchtechniques utilize DNA ligase to incorporate nucleotides and identifythe incorporation of such nucleotides. Example SBS systems and methodswhich can be utilized with the methods and systems described herein aredescribed in U.S. Pat. No. 6,969,488, U.S. Pat. No. 6,172,218, and U.S.Pat. No. 6,306,597, the disclosures of which are incorporated herein byreference in their entireties.

An additional example of a sequencing platform that can be used inassociation with the methods described herein is provided by CompleteGenomics Inc. (Mountain View, Calif.). Libraries of target nucleic acidscan be prepared where target nucleic acid sequences are interspersedapproximately every 20 bp with adaptor sequences. The target nucleicacids can be amplified using rolling circle replication, and theamplified target nucleic acids can be used to prepare an array of targetnucleic acids. Methods of sequencing such arrays include sequencing byligation, in particular, sequencing by combinatorial probe-anchorligation (cPAL).

In some embodiments using cPAL, about 10 contiguous bases adjacent to anadaptor may be determined. A pool of probes that includes four distinctlabels for each base (A, C, T, G) is used to read the positions adjacentto each adaptor. A separate pool is used to read each position. A poolof probes and an anchor specific to a particular adaptor is delivered tothe target nucleic acid in the presence of ligase. The anchor hybridizesto the adaptor, and a probe hybridizes to the target nucleic acidadjacent to the adaptor. The anchor and probe are ligated to oneanother. The hybridization is detected and the anchor-probe complex isremoved. A different anchor and pool of probes is delivered to thetarget nucleic acid in the presence of ligase. Preferably any apparatusand method of the present disclosure may be provided in an automatedform. The present application provides a solution to current andemerging needs that scientists and the biotechnology industry are tryingto address in the fields of genomics, pharmacogenomics, drug discovery,food characterization and genotyping, for example: in nucleic acidsequencing and re-sequencing, diagnostics and screening, gene expressionmonitoring, genetic diversity profiling, whole genome polymorphismdiscovery and scoring, the creation of genome slides (whole genome of apatient on a microscope slide) and whole genome sequencing.

A yet further aspect of the invention provides a kit for use insequencing, re-sequencing, gene expression monitoring, genetic diversityprofiling, diagnosis, screening, whole genome sequencing, whole genomepolymorphism discovery and scoring, or any other applications involvingthe amplification of nucleic acids or the sequencing thereof. This kitcomprises a plurality of nucleic acid clusters having identifiablecenters bound to a solid support, as outlined above.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

As used herein, in particular embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 5%. In other embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 10%. In yet other embodiments, the terms“about” or “approximately” when preceding a numerical value indicatesthe value plus or minus a range of 15%.

For example, in one embodiment, an oligonucleotide of approximately 20nucleotides in length is equivalent to oligonucleotides that range from19 to 21 nucleotides in length. In another embodiment, anoligonucleotide of approximately 20 nucleotides in length is equivalentto oligonucleotides that range from 18 to 22 nucleotides in length. Inyet another embodiment, an oligonucleotide of approximately 20nucleotides in length is equivalent to oligonucleotides that range from17 to 23 nucleotides in length.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are required and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements. As used herein, the term “each” when used in reference to acollection of items is intended to identify one or more individual itemsin the collection but does not necessarily refer to every item in thecollection unless the content clearly dictates otherwise.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of chemistry,biochemistry, organic chemistry, molecular biology, microbiology,recombinant DNA techniques, genetics, immunology, cell biology, stemcell protocols, cell culture and transgenic biology that are within theskill of the art, many of which are described below for the purpose ofillustration. Such techniques are explained fully in the literature.See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual(3^(rd) Edition, 2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual (2^(nd) Edition, 1989); Maniatis et al., MolecularCloning: A Laboratory Manual (1982); Ausubel et al., Current Protocolsin Molecular Biology (John Wiley and Sons, updated July 2008); ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-interscience; Glover, DNA Cloning: A Practical Approach, vol. I &II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis ofComplex Genomes, (Academic Press, New York, 1992); Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology (Academic Press, New York,1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, Eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R.Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); Fire et al., RNA Interference Technology: From Basic Science toDrug Development (Cambridge University Press, Cambridge, 2005);Schepers, RNA Interference in Practice (Wiley-VCH, 2005); Engelke, RNAInterference (RNAi): The Nuts & Bolts of siRNA Technology (DNA Press,2003); Gott, RNA Interference, Editing, and Modification: Methods andProtocols (Methods in Molecular Biology; Human Press, Totowa, N.J.,2004); Sohail, Gene Silencing by RNA Interference: Technology andApplication (CRC, 2004); Clarke and Sanseau, microRNA: Biology, Function& Expression (Nuts & Bolts series; DNA Press, 2006); Immobilized CellsAnd Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(J. H. Miller and M. P. Calos eds., 1987, Cold Spring HarborLaboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In CellAnd Molecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir andC C Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition,(Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells:Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen,Ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation andCharacterization (Methods in Molecular Biology) (Kurstad Turksen, Ed.,2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models(Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); HumanEmbryonic Stem Cell Protocols (Methods in Molecular Biology) (KursadTurksen Ed., 2006); Mesenchymal Stem Cells: Methods and Protocols(Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney,and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols(Methods in Molecular Medicine) (Christopher A. Klug, and Craig T.Jordan Eds., 2001); Hematopoietic Stem Cell Protocols (Methods inMolecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells:Methods and Protocols (Methods in Molecular Biology) (Leslie P. WeinerEd., 2008); Hogan et al., Methods of Manipulating the Mouse Embyro(2^(nd) Edition, 1994); Nagy et al., Methods of Manipulating the MouseEmbryo (3^(rd) Edition, 2002), and The zebrafish book. A guide for thelaboratory use of zebrafish (Danio rerio), 4th Ed., (Univ. of OregonPress, Eugene, Oreg., 2000).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1-20. (canceled)
 21. A method for producing at least one nucleic acid cluster having an identifiable center, comprising: (a) generating, on a solid support, at least one immobilized nucleic acid complement of at least one nucleic acid template; (b) carrying out a first set of amplification cycles to amplify the at least one nucleic acid template and the at least one nucleic acid complement in the presence of a nucleoside analog to obtain a region comprising immobilized copies of the at least one nucleic acid template and of the at least one nucleic acid complement, the immobilized copies comprising the nucleoside analog; (c) carrying out a second set of amplification cycles in the absence of the nucleoside analog to obtain on the solid support at least one nucleic acid cluster, wherein each cluster comprises (i) a substantially central location comprising the at least one nucleic acid template and the at least one nucleic acid complement comprising the nucleoside analog, and (ii) a surrounding region comprising one or more immobilized copies of the at least one nucleic acid template and of the at least one nucleic acid complement lacking the nucleoside analog; and (d) selectively removing nucleic acids in the substantially central location while leaving the nucleic acids in the surrounding region, thereby producing at least one nucleic acid cluster having an identifiable center.
 22. The method of claim 21, wherein selectively removing comprises treating the nucleoside analog to generate an abasic site capable of endonuclease cleavage.
 23. The method of claim 21, wherein the nucleoside analog comprises uracil.
 24. The method of claim 21, wherein the nucleoside analog comprises 8-oxo-guanine.
 25. The method of claim 21, further comprising labeling the surrounding region with a detectable label.
 26. The method of claim 25, wherein the labelling comprises binding an intercalator stain to the nucleic acids in the surrounding region.
 27. The method of claim 25, wherein labeling comprises hybridizing a sequencing primer to the immobilized copies of the at least one nucleic acid template or of the at least one nucleic acid complement and extending the primer with at least one fluorescently labeled nucleotide.
 28. The method of claim 25, further comprising detecting the detectable label in the surrounding region.
 29. The method of claim 25, wherein the resulting cluster comprises an outer ring comprising labeled nucleotides surrounding a dark central region.
 30. The method of claim 29, further comprising using the outer ring or the dark central region as a fiducial marking for performing an image registration.
 31. The method of claim 29, further comprising using the outer ring or the dark central region as a fiducial marking to focus an imaging device.
 32. The method of claim 21, wherein the solid support is contained in a flow cell.
 33. The method of claim 21, wherein the clusters comprise a circular shape.
 34. The method of claim 21, wherein the clusters comprise a shape other than a circular shape.
 35. The method of claim 22, wherein treating the nucleoside analog comprises digesting nucleic acids using a glycosylase.
 36. The method of claim 35, wherein the glycosylase comprises uracil DNA glycosylase.
 37. The method of claim 21, wherein the amplifying in (b) is isothermal amplification.
 38. The method of claim 21, wherein the amplifying in (b) is isothermal amplification.
 39. A solid support comprising a plurality of fiducial markings, the markings comprising a ring of fluorescently labeled nucleic acids surrounding a dark region.
 40. A method of image registration comprising imaging a solid support comprising a plurality of fiducial markings, the markings comprising a ring of fluorescently labeled nucleic acids surrounding a dark region to identify the location of nucleic acid clusters in multiple images. 